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Subjective Questions

Q.1- Discuss the metabolism of Galactose and explain how does galactose gain entry in to the glycolytic pathway?

 Answer- Galactose is derived from intestinal hydrolysis of the disaccharide lactose, the sugar of milk. It is readily converted in the liver to glucose.

 

Figure 1- showing the metabolism of galactose. Classical galactosemia occurs due to deficiency of Galactose-1-P uridyl transferase as shown by the block

Metabolism of Galactose

1) Step 1- Formation of galactose-1-P – Galactokinase catalyses the phosphorylation of galactose, using ATP as phosphate donor.(Figure 1)

 2) Step 2 -Epimerization of galactose-1-phosphate to Glucose-1-P (G1P) requires the transfer of UDP from uridine diphosphoglucose (UDP-glucose) catalyzed by galactose-1-phosphate uridyl transferase. This generates UDP-galactose and G1P.

 3) Step 3- Epimerization of UDP galactose to UDP Glucose- The UDP-galactose is epimerized to UDP-glucose by UDP-galactose-4 epimerase. The reaction involves oxidation, then reduction, at carbon 4, with NAD+ as coenzyme.

Fate of UDP glucose-  i) The UDPGlc can be incorporated into glycogen.

ii) Since the epimerase reaction is freely reversible, UDP glucose can be converted to UDP galactose, so that galactose is not a dietary essential.

iii) UDPGlc can also be used in the Uronic acid pathway for the formation of UDP glucuronic acid which can be utilized for the conjugation reactions.

Fate of glucose-1-P- Glucose-1-phosphate can be converted to G6P by phosphoglucose mutase. Glucose -6-P can then enter glycolysis (Figure 2) or can be converted to free glucose by the action of glucose-6 phosphatase.

 

Figure-2 showing the entry of galactose in to glycolytic pathway

Fate of UDP galactose

UDP Galactose supplies galactose moiety for the formation of lactose, glycolipids (Cerebrosides), Proteoglycans, and glycoproteins.

Biosynthesis of Galacitol

Galacitol is an alcohol that is made from galactose. The pathway is similar to that of formation of glucose. Because of the excessive quantity of galactose, some of it is converted to galacitol, which build up in the body and is excessively excreted in urine. Galacitol appears to be toxic at high concentration and produces tissue injuries, especially cataract formation.(see the details in galactosemia )

Q.2- A patient complains of gastric discomfort upon consuming milk. The patient also shows signs of liver, kidney and brain dysfunction. This dysfunction is due to the build up of the toxic compound, galactose-1-P.

  • What enzyme is the patient lacking?
  • What reaction is not being catalyzed?
  • What kind of laboratory investigations should be carried out for the confirmation of diagnosis?

Suggest a possible recommendation for the patient to follow in order to avoid further tissue damage.

Answer- The patient might be suffering from Classical Galactosemia as is evident from the signs and symptoms and the accumulation of Galactose-1-P.

Galactose-1-phosphate uridyl Transferase (GALT) deficiency is the most common enzyme deficiency that causes Galactosemia.

Galactose-1-phosphate uridyl Transferase (GALT) catalyzes conversion of galactose-1-p to UDP Galactose. This is an important reaction during the metabolism of galactose,

Galactosemia

Galactosemia is associated with the following 3 enzyme deficiencies;

A) Classical galactosemia is a major symptom of two enzyme defects. It results from loss of the enzyme galactose-1-phosphate uridyl transferase.

B) The second form of galactosemia results from a loss of galactokinase.

Clinical manifestations- These two defects are manifested by-

1) A failure of neonates to thrive.

2)Vomiting and diarrhea occur following ingestion of milk; hence individuals are termed lactose intolerant.

3)Impaired liver function (which if left untreated leads to severe cirrhosis),

4) Elevated blood galactose, hypergalactosemia, hyperchloremic metabolic acidosis, urinary galacitol excretion and hyper aminoaciduria. Unless controlled by exclusion of galactose from the diet, these galactosemias can go on to produce blindness and fatal liver damage. Even on a galactose-restricted diet, transferase-deficient individuals exhibit urinary galacitol excretion and persistently elevated erythrocyte galactose-1-phosphate levels. B

5) Blindness is due to the conversion of circulating galactose to the sugar alcohol galacitol, by an NADPH-dependent galactose reductase that is present in neural tissue and in the lens of the eye. At normal circulating levels of galactose this enzyme activity causes no pathological effects. However, a high concentration of galacitol in the lens causes osmotic swelling, with the resultant formation of cataracts and other symptoms. The principal treatment of these disorders is to eliminate lactose from the diet.

6) Hypoglycemia is a very common finding due to inhibition of Phosphoglucomutase enzyme.

7)Mental retardation occurs in the untreated cases due to accumulation of galactose and galactose-1-P.

C) The third disorder results from a deficiency of UDP-galactose-4-epimerase-Two different forms of this deficiency have been found. One is benign affecting only red and white blood cells. The other affects multiple tissues and manifests symptoms similar to the transferase deficiency. Treatment involves restriction of dietary galactose.

Laboratory Investigations

I) Urine test for reducing sugar is positive. The confirmation for the presence of galactose can be done by Thin Layer Chromatography.

II) Galactose Tolerance Test- A galactose Tolerance Test is abnormal in these patients. There is much higher than normal rise in blood galactose after the administration of galactose, and the elevation persists for a much longer time than normal. The galactose Tolerance Test in a normal person shows the level in the blood rising to a maximum at about half an hour after the galactose administration. It then returns to base line value within 1 hour.

III) Serum Transaminases- Both the serum Transaminases are elevated, suggesting the possibility of liver damage.

IV) Serum Bilirubin- is elevated. A high proportion of the bilirubin is unconjugated.

V)  Estimation of Galactose-1-phosphate Uridyl Transferase- Finally the diagnosis can be made by demonstrating that the galatose-1-phosphate Transferase is absent in the erythrocytes.

Treatment- Galactosemic children are to be kept on a galactose free diet. Synthetic diets or soya milk can be substituted.

Q.3- Explain why a galactosemic child maintained on a galactose free diet manifests normal growth and development? What is the alternative pathway for the formation of galactose in the body?

Answer- Galactose is required in the brain for the synthesis of glycolipids like Cerebrosides and gangliosides. It is essential for the synthesis of glycoproteins like membrane proteins and protein hormones.

Galactosemia is treated by giving a galactose free diet. In the absence of dietary galactose the patient can thrive, an alternative source of galactose exists even if the patient is fed on a galactose free diet because the much needed galactose for the synthesis of biomolecules comes from UDP glucose by the action of Epimerase enzyme (Figure 1). First, glucose is converted to glucose-1-phosphate and then to UDP- glucose. Next an Epimerase converts the UDP- glucose to UDP- galactose. Hence a child on a galactose free diet can have a normal growth and development at the cost of glucose compensating for galactose.

Patients with Transferase deficiency, eventually have a less severe disease because an alternative enzyme for galactose utilization develops during childhood. This enzyme enables the galactose-1-phosphate to bypass the uridyl Transferase step. Instead of reacting with UDP –glucose, the galactose-1-phosphate reacts with UTP to form UDP galactose. The alternative pathway can handle only a fraction of the galactose that the uridyl Transferase reaction can. It handles enough, however, to enable the person to have some galactose in the diet. Therefore a patient does not have to maintain as severely a restrictive diet as he or she grows older. If the Galactokinase were deficient alternate pathway could not function later in life because the patient could not produce galactose -1 phosphate in the diet. Therefore the patient would have to remain on a much more restrictive diet throughout life. Fortunately very few cases of galactosemia are caused by Galactokinase deficiency.

Q.4- A pregnant woman who was extremely lactose intolerant asked her physician if she could still be able to breast feed her baby even though she could not drink milk or dairy products. What advice should be given?

Answer- The patient should be advised to breast feed the baby after delivery. Lactose is synthesized from UDP galactose and glucose; however galactose is not required in the diet for lactose synthesis, because galactose can be synthesized from glucose.

Lactose synthesis

Lactose is unique in the sense that it can only be synthesized in the mammary gland of the adult female for short period during lactation. Lactose synthase present in the endoplasmic reticulum of the lactating mammary gland, catalyze the last step in the lactose biosynthesis, the transfer of galactose from UDP galactose to glucose.

 

Figure 3- showing the formation of lactose, dietary galactose is not needed for lactose synthesis. Glucose is converted first to Glucose-6-p, then to Glucose-1-p, which is converted to UDP glucose and then to UDP galactose to condense with glucose to form lactose.

Lactose synthase attaches the anomeric carbon of galactose to C4 alcohol group of glucose to form a glycosidic bond. Lactose synthase is composed of a galactosyl transferase and α lactalbumin which is a regulatory subunit. Lactose synthase is activated by Prolactin hormone. In the non lactating mammary glands, this enzyme is inactive, so lactose is not synthesized and UDP galactose is alternatively used for the formation of glycolipids or glycoproteins.

 Q.5- High concentration of galactose-1-phosphate inhibits Phosphoglucomutase, the enzyme that converts glucose-6-P to Glucose-1-P. How can this inhibition account for hypoglycemia and jaundice that accompany galactose-1-P uridyl transferase deficiency?

Answer- Inhibition of Phosphoglucomutase by galactose-1-P results in hypoglycemia due to interference in formation of UDP glucose (Glycogen precursor) and also in the degradation of glycogen back to glucose-6-p.

 90% of glycogen is converted to Glucose-1-p which is converted to glucose-6-p by Phosphoglucomutase enzyme. When Phosphoglucomutase is inhibited less glucose-6-p is formed and hence less free glucose is formed to be exported. Thus stored glycogen is only 10% efficient in raising blood glucose level and hence hypoglycemia results.

UDP glucose levels are reduced, because glucose-1-p is required for the formation of UDP glucose. Hence in the absence of Phosphoglucomutase activity, glucose-6-p (derived from the activity of glucokinase or from gluconeogenesis), can not be converted to glucose-1-p. This prevents the formation of UDP glucuronic acid which is required to convert bilirubin to bilirubin diglucuronide form for transport in to bile. Bilirubin accumulates in tissues causing jaundice. 

Besides conjugation, the uptake of bilirubin in hepatocytes is also affected resulting in unconjugated type of hyperbilirubunemia.

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Q.1- Why do animals store energy as glycogen? Why not convert all excess fuel into fatty acids?

Answer- Glycogen is a readily mobilized storage form of glucose. It is a very large, branched polymer of glucose residues that can be broken down to yield glucose molecules when energy is needed. Most of the glucose residues in glycogen are linked by α-1,4-glycosidic bonds. Branches at about every tenth residue are created by α-1,6-glycosidic bonds (Figure 1).

 

Figure 1- Showing glycogen structure, the non reducing ends have been shown. R – Represents the remaining structure of glycogen molecule

Glycogen is not as reduced as fatty acids are and consequently not as energy rich. Glycogen is an important fuel reserve for several reasons-

 1) The controlled breakdown of glycogen and release of glucose increase the amount of glucose that is available between meals. Hence, glycogen serves as a buffer to maintain blood-glucose levels.

2) Glycogen’s role in maintaining blood glucose levels is especially important because glucose is virtually the only fuel used by the brain, except during prolonged starvation.

3) The glucose from glycogen is readily mobilized and is therefore a good source of energy for sudden, strenuous activity.

4) Unlike fatty acids, the released glucose can provide energy in the absence of oxygen and can thus supply energy for anaerobic activity.

The two major sites of glycogen storage are the liver and skeletal muscle. The concentration of glycogen is higher in the liver than in muscle (10% versus 2% by weight), but more glycogen is stored in skeletal muscle overall because of its much greater mass. Glycogen is present in the cytosol in the form of granules ranging in diameter from 10 to 40 nm. In the liver, glycogen synthesis and degradation are regulated to maintain blood-glucose levels as required to meet the needs of the organism as a whole. In contrast, in muscle, these processes are regulated to meet the energy needs of the muscle itself.

Q.2- What is Glycogenesis? Describe the steps and state under what conditions glycogenesis would be promoted in the body?

Or

Describe the separate roles of Glycogenin and Glycogen Synthase in glycogen synthesis. Summarize the reactions catalyzed by each enzyme.

Answer- Glycogenesis is the synthesis of glycogen from glucose. Glycogenesis mainly occurs in muscle and liver. Muscle glycogen provides a readily available source of glucose for glycolysis within the muscle itself. Liver glycogen functions to store and export glucose to maintain blood glucose between meals. After 12–18 hours of fasting, liver glycogen is almost totally depleted. Although muscle glycogen does not directly yield free glucose, (because muscle lacks glucose 6-phosphatase), pyruvate formed by glycolysis in muscle can undergo transamination to alanine, which is exported from muscle and used for gluconeogenesis in the liver.

Steps of Glycogen Synthesis

1) Activation of Glucose

Synthesis of glycogen from glucose is carried out by the enzyme glycogen synthase. This enzyme utilizes UDP-glucose as one substrate and the non-reducing end of glycogen as another. UDP-glucose, the glucose donor in the biosynthesis of glycogen, is an activated form of glucose, just as ATP and acetyl CoA are activated forms of orthophosphate and acetate respectively.

As in glycolysis, glucose is phosphorylated to glucose 6-phosphate, catalyzed by hexokinase in muscle and glucokinase in liver (Figure 2). Glucose 6-phosphate is isomerized to glucose 1-phosphate by Phosphoglucomutase. The enzyme itself is phosphorylated, and the Phospho-group takes part in a reversible reaction in which glucose 1,6-bisphosphate is an intermediate. Next, glucose 1-phosphate reacts with uridine triphosphate (UTP) to form the active nucleotide uridine diphosphate glucose (UDPGlc) and pyrophosphate (Figure 3). The reaction is catalyzed by UDPGlc pyro phosphorylase. The reaction proceeds in the direction of UDPGlc formation because pyrophosphatase catalyzes hydrolysis of pyrophosphate to 2 x phosphate, so removing one of the reaction products.

 

Figure 2- showing activation of glucose to UDP glucose and its incorporation in to the pre- existing glycogen fragment

 

Figure-3- Showing formation of UDP glucose, The C-1 carbon atom of the glucosyl unit of UDP glucose is activated because its hydroxyl group is esterified to the diphosphate moiety of UDP.

The essentially irreversible hydrolysis of pyrophosphate drives the synthesis of UDP-glucose.

 

2) Initiation- Glycogen synthase can add glucosyl residues only if the polysaccharide chain already contains more than four residues. Thus, glycogen synthesis requires a primer. This priming function is carried out by glycogenin, a protein composed of two identical 37-kd subunits, each bearing an oligosaccharide of alpha-1,4-glucose units (Figure-4).

Figure 4- showing diagrammatic representation of glycogenin dimer

Carbon 1 of the first unit of this chain, the reducing end, is covalently attached to the phenolic hydroxyl group of a specific tyrosine in each glycogenin subunit (Figure 5). Each subunit of glycogenin catalyzes the addition of eight glucose units to its partner in the glycogenin dimer. UDP-glucose is the donor in this autoglycosylation. At this point, glycogen synthase takes over to extend the glycogen molecule. In skeletal muscle, glycogenin remains attached in the center of the glycogen molecule (Figure-7) ; in liver the number of glycogen molecules is greater than the number of glycogenin molecules.

 

 

Figure 5- A glycosidic bond is formed between the anomeric C1 of the glucose moiety derived  from UDP-glucose and the hydroxyl oxygen of a tyrosine side-chain of Glycogenin. UDP is  released as a product.

3)  Elongation - New glucosyl units are added to the nonreducing terminal residues of glycogen. The activated glucosyl unit of UDPglucose is transferred to the hydroxyl group at a C-4 terminus of glycogen to form an α-1,4-glycosidic linkage. In elongation, UDP is displaced by the terminal hydroxyl group of the growing glycogen molecule. This reaction is catalyzed by glycogen synthase, the key regulatory enzyme in glycogen synthesis.

4) Glycogen branching- Glycogen synthase catalyzes only the synthesis of α-1,4 linkages. Another enzyme is required to form the α-1,6 linkages that make glycogen a branched polymer. Branching occurs after a number of glucosyl residues are joined in α-1,4 linkage by glycogen synthase. A branch is created by the breaking of an α-1,4 link and the formation of an α–1,6 link: this reaction is different from debranching. A block of residues, typically 7 in number, is transferred to a more interior site. The branching enzyme that catalyzes this reaction is quite exacting. The block of 7 or so residues must include the nonreducing terminus and come from a chain at least 11 residues long. In addition, the new branch point must be at least 4 residues away from a preexisting one. Branching is important because it increases the solubility of glycogen. Furthermore, branching creates a large number of terminal residues, the sites of action of glycogen phosphorylase and synthase (Figure 6). Thus, branching increases the rate of glycogen synthesis and degradation.

                                 

 Figure-6- showing the transfer of 7 glucosyl residue by the branching enzyme to create a branch point.                                               

 

Figure 7- Showing structure of glycogen, The glycogen molecule is a sphere approximately 10-40 nm in diameter that can be seen in electron micrographs. It has a molecular mass of 107 Da and consists of polysaccharide chains, each containing about 13 glucose residues. The chains are either branched or unbranched and are arranged in 12 concentric layers .The branched chains (each has two branches) are found in the inner layers and the unbranched chains in the outer layer. (G, glycogenin, the primer molecule for glycogen synthesis.)

Glycogen Synthase, The key regulatory enzyme of Glycogen synthesis is activated by glucose-6-phosphate. Thus Glycogen Synthase is active when high blood glucose leads to elevated intra cellular glucose-6-phosphate.

It is useful to a cell to store glucose as glycogen when the input to Glycolysis (glucose-6-phosphate), and the main product of Glycolysis (ATP), are adequate.

Q.3- Explain the pathway by which Glycogen is degraded in the body?

Answer- Glycogenolysis (degradation of glycogen) is not just a reverse of glycogenesis, it is a separate pathway. Glycogen degradation consists of three steps: (1) the release of glucose 1-phosphate from glycogen, (2) the remodeling of the glycogen substrate to permit further degradation, and (3) the conversion of glucose 1-phosphate into glucose 6-phosphate for further metabolism.

Enzymes required for glycogen degradation

The efficient breakdown of glycogen to provide glucose 6-phosphate for further metabolism requires four enzyme activities: one to degrade glycogen, two to remodel glycogen so that it remains a substrate for degradation, and one to convert the product of glycogen breakdown into a form suitable for further metabolism.

a) Phosphorylase- Glycogen phosphorylase, the key enzyme in glycogen breakdown, cleaves its substrate by the addition of orthophosphate (Pi) to yield glucose 1-phosphate. The cleavage of a bond by the addition of orthophosphate is referred to as phosphorolysis. 

b) Transferase and Debranching enzyme- The transferase shifts a block of three glucosyl residues from one outer branch to the other. This transfer exposes a single glucose residue joined by an alpha-1,6-glycosidic linkage. alpha-1,6-Glucosidase, also known as the debranching enzyme, hydrolyzes the alpha-1, 6-glycosidic bond, resulting in the release of a free glucose molecule.

c) Phosphoglucomutase- Glucose 1-phosphate formed in the phosphoroylytic cleavage of glycogen must be converted into glucose 6-phosphate to enter the metabolic mainstream. This shift of a phosphoryl group is catalyzed by Phosphoglucomutase.

 Steps of Glycogen degradation

 1) Release of Glucose-1-P- Phosphorylase catalyzes the sequential removal of glucosyl residues from the nonreducing ends of the glycogen molecule (the ends with a free 4-OH group. Orthophosphate splits the glycosidic linkage between C-1 of the terminal residue and C-4 of the adjacent on (Figure-8). Specifically, it cleaves the bond between the C-1 carbon atom and the glycosidic oxygen atom, and the a configuration at C-1 is retained.

 

 

 

Figure 8- Showing the release of Glucose-1-P by the action of Phosphorylase enzyme

Glucose 1-phosphate released from glycogen can be readily converted into glucose 6-phosphate an important metabolic intermediate, by the enzyme Phosphoglucomutase.

Advantages of phosphoroylytic cleavage- The phosphoroylytic cleavage of glycogen is energetically advantageous because the released sugar is already phosphorylated. In contrast, a hydrolytic cleavage would yield glucose, which would then have to be phosphorylated at the expense of the hydrolysis of a molecule of ATP to enter the glycolytic pathway. An additional advantage of phosphoroylytic cleavage for muscle cells is that glucose 1-phosphate, negatively charged under physiological conditions, cannot diffuse out of the cell.

 2) Remodeling of the glycogen substrate to permit further degradation- The alpha-1,6-glycosidic bonds at the branch points are not susceptible to cleavage by phosphorylase. Glycogen phosphorylase stops cleaving alpha-1,4 linkages when it reaches a terminal residue four residues away from a branch point (figure-9). Because about 1 in 10 residues is branched, glycogen degradation by the phosphorylase alone would come to a halt after the release of six glucose molecules per branch.

 

 

 

Figure 9- Remodeling of glycogen structure by Transferase and debranching enzyme, the transferase shifts a block of three glycosyl residues from one outer branch to the other. This transfer exposes a single glucose residue joined by an a-1,6-glycosidic linkage. a-1,6-Glucosidase, also known as the debranching enzyme, hydrolyzes the a-1, 6-glycosidic bond, resulting in the release of a free glucose molecule.

 Transferase and a-1,6-glucosidase, remodel the glycogen for continued degradation by the phosphorylase. The free glucose molecule (Figure-10)released by the action of debranching enzyme is phosphorylated by the glycolytic enzyme hexokinase. Thus, the transferase and alpha-1,6-glucosidase convert the branched structure into a linear one, which paves the way for further cleavage by phosphorylase.

In eukaryotes, the transferase and the a-1,6-glucosidase activities are present in a single 160-kd polypeptide chain, providing yet another example of a bifunctional enzyme

 

 

Figure 10- showing the action of debranching enzyme, free glucose is released by the action of debranching enzyme

3) Conversion of glucose 1-phosphate into glucose 6-phosphate for further metabolism

Phosphoglucomutase converts glucose 1-phosphate into glucose 6-phosphate in a reversible reaction. The catalytic site of an active mutase molecule contains a phosphorylated serine residue. The phosphoryl group is transferred from the serine residue to the C-6 hydroxyl group of glucose 1-phosphate to form glucose 1,6-bisphosphate. The C-1 phosphoryl group of this intermediate is then shuttled to the same serine residue, resulting in the formation of glucose 6-phosphate and the regeneration of the phosphoenzyme.

Glucose 6-phosphate derived from glycogen can (1) be used as a fuel for anaerobic or aerobic metabolism as in, for instance, muscle; (2) be converted into free glucose in the liver and subsequently released into the blood; (3) be processed by the pentose phosphate pathway to generate NADPH or ribose in a variety of tissues (Figure-11)

 

Figure 11- showing the fate of glucose-6-P

Conversion of Glucose-6-P to free Glucose

The liver contains a hydrolytic enzyme, glucose 6-phosphatase, which cleaves the phosphoryl group to form free glucose and orthophosphate.

Glucose 6-phosphatase is absent from most other tissues. Consequently, glucose 6-phosphate is retained for the generation of ATP. In contrast, glucose is not a major fuel for the liver. The liver releases glucose into the blood during muscular activity and between meals to be taken up primarily by the brain and skeletal muscle.

 Q.4- What is the role played by pyridoxal phosphate in glycogen metabolism?

Answer- Pyridoxal phosphate (PLP), a derivative of vitamin B6, serves as prosthetic group for Glycogen Phosphorylase. 

Pyridoxal phosphate is held at the active site of Phosphorylase enzyme by a Schiff base linkage, formed by reaction of the aldehyde of PLP with the e-amino group of a lysine residue (Figure-12).

In contrast to the role of this cofactor in other, the phosphate moiety of PLP is involved in acid/base catalysis by Phosphorylase. 

The Pi substrate binds between the phosphate of PLP and the glycosidic oxygen linking the terminal glucose residue of the glycogen substrate.

After the phosphate substrate donates a proton during cleavage of the glycosidic bond, it receives a proton from the phosphate moiety of PLP. PLP then takes back the proton as the phosphate oxygen attacks C1 of the cleaved glucose to yield glucose-1-phosphate.

 

Figure-12- showing Schiff base linkage A pyridoxal phosphate group (red) forms a Schiff base with a lysine residue (blue) at the active site of phosphorylase.

Q.5- What is the cost of converting glucose 6-phosphate into glycogen and back into glucose 6-phosphate?

Or

Q.6-Is the energy required to synthesize glycogen from glucose 6-phosphate the same as the energy required to degrade glycogen to glucose 6-phosphate?

Answer- No. More energy is required to synthesize glycogen from glucose 6-P.

The details of energy expenditure per reaction are as follows-

 

Thus, one ATP is hydrolyzed incorporating glucose 6-phosphate into glycogen. The energy yield from the breakdown of glycogen is highly efficient. About 90% of the residues are phosphorolytically cleaved to glucose 1-phosphate, which is converted at no cost into glucose 6-phosphate. The other 10% are branch residues, which are hydrolytically cleaved. One molecule of ATP is then used to phosphorylate each of these glucose molecules to glucose 6-phosphate.

Q.7-The phosphoroylytic cleavage of glycogen is key for glycogen metabolism. Why?

Answer- Hydrolysis of glycogen will generate glucose which is free to leave the cell and will require the additional input of energy to phosphorylate to glucose 6-P. Phosphoroylytic cleavage of glycogen produces glucose 1-P which can be converted to glucose 6 P and be utilized by several different pathways. This reaction does not require ATP and is therefore more efficient because it decreases the ATP investment.

Q.8-Why is it important to have different pathways for glycogenesis and glycogenolysis in liver and muscle cells?

Answer- The separate pathways for the synthesis and degradation of glycogen allow the synthesis of glycogen to proceed despite a high ratio of Pi to glucose 1 phosphate. The separate pathways allow the coordinated reciprocal control of glycogen synthesis and degradation by hormonal regulation.

 

 

 

 

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Q.1 -Discuss the steps of Uronic acid pathway, what is the biological significance of this pathway?

 Answer- The uronic acid pathway is an alternative pathway for the oxidation of glucose that does not provide a means of producing ATP, but is utilized for the generation of the activated form of glucuronate, UDP-glucuronate which is mainly used for detoxification of foreign chemicals and for the synthesis of Mucopolysaccharides. This pathway also produces Ascorbic acid in certain animals.

 The unutilized Glucuronate produced in this pathway is converted to Xylulose-5 P which is further metabolized through HMP pathway

Steps of Uronic acid pathway

 

 

 

 

 

 

 

 

 

 

 

 

Figure-1- showing the reactions of Uronic acid pathway

 1) Formation of UDP glucose

Glucose 6-phosphate is isomerized to glucose 1-phosphate in a reaction catalyzed by Phosphoglucomutase, which then reacts with uridine triphosphate (UTP) to form uridine diphosphate glucose (UDPGlc) in a reaction catalyzed by UDPGlc pyro phosphorylase, as occurs in glycogen synthesis. (Figure-1)

2) Formation of D- Glucuronic acid

UDPGlc is oxidized at carbon 6 by NAD-dependent UDPGlc dehydrogenase in a two-step reaction to yield UDP-glucuronate. (Figure 1)

UDP-glucuronate is the source of glucuronate for reactions involving its incorporation into Proteoglycans or for reactions of substrates such as steroid hormones, bilirubin, and a number of drugs that are excreted in urine or bile as glucuronide conjugates. UDP- G is hydrolyzed to form D- Glucuronic acid.

3) Formation of L- Gulonic acid

Glucuronate is first reduced by the NADPH dependent enzyme, Glucuronate reductase to form L- gulonate which is dehydrated in the presence of enzyme, Aldonolactonase to form L-gulono-δ-lactone, the direct precursor of ascorbate in those animals capable of synthesizing this vitamin, in an NADPH-dependent reaction. Removal of a pair of hydrogen atoms from L-gulono-δ-lactone, under the effect of enzyme L-gluconolactone oxidase leads to the formation of 2-keto gulono lactone which is finally converted to L ascorbic acid. (Figure-2)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2- showing the synthesis of ascorbic acid from D- glucuronic acid. The enzyme L- gluconolactone oxidase is absent in human beings and in certain animals as shown by the block at the step. D- Glucuronic acid in such species is converted to Xylulose-5-p through a number of steps, which enters Pentose phosphate pathway for further metabolism.

This pathway is used by plants and some animals for the synthesis of Ascorbic acid. In humans and other primates, as well as in guinea pigs, bats, and some birds and fishes, ascorbic acid cannot be synthesized because of the absence of L-gluconolactone oxidase. It is due to genetic deficiency of this enzyme. It appears that the capacity to synthesize ascorbic acid was lost in these species due to a mutation which was not lethal. These species require vitamin C in the diet. Thus a single enzyme defect in the Uronic acid pathway is responsible for inefficiency to synthesize ascorbic acid in primates.

4) Fate of L- Gulonate

Uronic acid pathway is connected to Pentose phosphate pathway through L-gulonate, since the latter can be converted to an intermediate of the Pentose phosphate pathway as follows-

 

 

 

 

 

L-Gulonate is oxidized to 3-keto-L-gulonate, which is then decarboxylated to L-Xylulose. L-Xylulose is converted to the D isomer by an NADPH-dependent reduction to xylitol, followed by oxidation in an NAD-dependent reaction to D-Xylulose. D- Xylulose is converted to converted to D-Xylulose 5-phosphate at the expense of ATP which is metabolized via the pentose phosphate pathway.(Figure-3)

 

 

 

 

 

 

 

 

 

 

 

Figure- 3- showing the fate of L- Xylulose

Biological significance of Uronic acid pathway- UDP glucuronate the active form of glucuronic acid, can readily donate the glucuronic acid component for the following functions-

1) Detoxification of foreign compounds and drugs- During detoxification, the glucuronate residues are covalently attached to these substances. Since glucuronate residues are strongly polar, their attachment imparts polar character to these substances, making them water-soluble and readily excretable. Bilirubin, certain hormones and drugs are made more polar for renal excretion in this manner.

2) Synthesis of Mucopolysaccharides-such as hyaluronic acid and heparin, which contain glucuronic acid as essential component.

 Q.2- What is the defect in essential Pentosuria? How can this be diagnosed? What are the clinical manifestations and how is this defect treated?

Or

A 35-year-old Lebanese Christian male was referred to the biochemical laboratory for the study of his mellituria. The urinary sugar had been discovered during a hospitalization for the repair of an inguinal hernia when the patient was 20 years of age, and he was then refused operation because of “diabetes.” Numerous blood sugar analyses were reported normal. The patient was in excellent health and had no symptoms of diabetes, but was concerned about his condition. L- xylulose was present excessively in urine. What is the probable diagnosis?

Answer- Pentosuria is the condition in which an unusual reducing substance, one of the pentose sugars, is constantly excreted in the urine and gives a positive reaction on testing with Benedict’s solution. It is a rare hereditary disease which has been included by Garrod (1923) among the inborn errors of metabolism. Its occurrence was first described in 1892, but since then only about 200 cases have been recorded in the literature, the disorder occurred almost entirely in the Jewish race.

Biochemical defect- The enzyme that causes conversion of L-Xylulose to Xylitol is deficient. As a result the excess of L-Xylulose is excreted in urine.

Clinical Manifestations- It may go unnoticed or it may be a chance finding on routine examination of urine. There are no signs and symptoms associated with it. Various drugs increase the rate at which glucose enters the uronic acid pathway. For example, administration of barbital or chlorobutanol to rats results in a significant increase in the conversion of glucose to glucuronate, L-gulonate, and ascorbate. Aminopyrine and antipyrine increase the excretion of L-xylulose in pentosuric subjects.

Diagnosis-It can be misdiagnosed with renal glycosuria or mild diabetes mellitus. The Qualitative Benedict’s test for reducing substances is given positive in this condition. Bial’s test and fasting blood glucose estimation can rule out renal glycosuria and diabetes mellitus.

The identification of urinary xylulose has been greatly facilitated by the introduction of paper chromatography.

Treatment- No treatment is required for this defect.

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Q.5 -Can fructose be converted to glucose or “blood sugar”? 

Answer- Fructose does not stimulate the release of insulin. The reduced insulin/glucagon ratio stimulates gluconeogenesis and inhibits glycolysis. That is, glucagon dominates the picture, increasing fructose bisphosphatase activity and leading to formation of glucose. Gluconeogenesis occurs only if fructose in pure form is consumed. However, the more usual situation is consumption of fructose as sugar as a sweetener in a “normal” meal.  In other words, fructose is consumed together with starch or sugar. This leads to increases in blood sugar and insulin levels directly with a rapid cessation of gluconeogenesis. 

Mechanism – Increased concentrations of DHAP (dihydroxy acetone phosphate)and glyceraldehyde 3-phosphate produced from the metabolism of fructose in the liver drive the pathway toward glucose and subsequent glycogen synthesis.

Dihydroxyacetone phosphate and Glyceraldehyde -3-P produced from fructose metabolism condense together in the presence of Aldolase to form Fructose 1,6 bisphosphate, that  is cleaved by fructose 1,6 bisphosphatase to form fructose-6-P. Glucose-6-P is produced from Fructose-6-P by the action of Phospho hexose isomerase and free glucose is produced from glucose-6-p by the action of glucose-6-phosphatse. Hence by these reactions, Glucose is produced from fructose and contributes to blood sugar level.

Surplus glucose-6-p can enter the pentose phosphate pathway or it can be converted to Glucose-1-P under the effect of Phosphoglucomutase enzyme, which is subsequently used up for the formation of Glycogen. (Figure 7)

It appears that fructose is a better substrate for glycogen synthesis than glucose and that glycogen replenishment takes precedence over triglyceride formation. Once liver glycogen is replenished, the intermediates of fructose metabolism are primarily directed toward triglyceride synthesis.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure -7- showing effects of fructose metabolism on glycogen synthesis.

Q.6– What is Essential fructosuria? 

Answer- Essential fructosuria, also known as hepatic fructokinase deficiency or keto hexokinase deficiency, is a hereditary metabolic disorder caused by a deficiency of hepatic fructokinase, leading to fructose being excreted in urine. The inheritance is autosomal recessive.

Essential fructosuria, apparently a harmless condition, is an extremely rare error of metabolism, the recognition of which is of importance because it may be mistaken for diabetes mellitus.It is characterized by the patient’s inability to utilize fructose normally, whether it is ingested as simple fructose or as a substance capable of yielding fructose on digestion, such as cane sugar. It is manifested clinically by a symptomless excretion of fructose in urine.

Essential fructosuria should not be confused with hereditary fructose intolerance which is a very serious condition, and is due to deficiency of Aldolase B enzyme. It causes a rise in uric acid, growth abnormalities, in severe cases hepatic or renal failure and finally coma or death.

On the other hand, being symptomless, fructosuria is commonly left undetected or undiagnosed.

Q.7– What is the reason that even after consuming a large amount of fructose rich drink, appetite is not suppressed?

Answer- Fructose does not stimulate release of insulin. One of insulin’s important functions is central regulation of hunger.  Fructose does not affect the hypothalamus directly or through insulin. Fructose does not appear to dampen the sense of hunger. 

Insulin release can modulate food intake by at least 2 mechanisms. First, insulin concentrations in the central nervous system have a direct inhibitory effect on food intake. In addition, insulin may modify food intake by its effect on leptin secretion, which is mainly regulated by insulin-induced changes in glucose metabolism in fat cells. Insulin increases leptin release with a time delay of several hours. Thus, a low insulin concentration after ingestion of fructose would be associated with lower average leptin concentrations than would be seen after ingestion of glucose. Because leptin inhibits food intake, the lower leptin concentrations induced by fructose would tend to enhance food intake and thus the appetite is not suppressed.

Q.8- What are the important differences between metabolism of glucose and fructose?

Answer- Fructose and Glucose differ from each other in their metabolism as follows-

1) Absorption -
The digestive and absorptive processes for glucose and fructose are different. When disaccharides such as sucrose or maltose enter the intestine, they are cleaved by disaccharidases. A sodium-glucose co transporter absorbs the glucose that is formed from cleavage of sucrose. Fructose, in contrast, is absorbed further down in the duodenum and jejunum by a non-sodium-dependent process. After absorption, glucose and fructose enter the portal circulation and either is transported to the liver, where  fructose can be taken up and converted to glucose, or passes into the general circulation.

2) Insulin release

Along with 2 peptides, glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 released from the gastrointestinal tract, circulating glucose increases insulin release from the pancreas. Fructose does not stimulate insulin secretion, probably because the ß cells of the pancreas lack the fructose transporter Glut-5.

3) Fructose metabolism

The metabolism of fructose differs from that of glucose in several other ways as  well; Glucose enters cells by a transport mechanism (Glut-4) that is insulin dependent in most tissues (Adipose tissues and skeletal muscles). Insulin activates the insulin receptor, which in turn increases the density of glucose transporters on the cell surface and thus facilitates the entry of glucose. Once inside the cell,glucose is phosphorylated by glucokinase/hexokinase to become glucose-6- phosphate, from which the intracellular metabolism of glucose begins. Intracellular enzymes can tightly control conversion of glucose-6-phosphate to the  glycerol backbone of triacylglycerols through modulation by phosphofructokinase.

In contrast with glucose, fructose enters cells via a Glut-5  transporter that does not depend on insulin. This transporter is absent from  pancreatic ß cells and the brain, which indicates limited entry of fructose into these tissues. Glucose provides “satiety” signals to the brain that fructose  cannot  provide because it is not transported into the brain. Once inside the cell, fructose is phosphorylated to form fructose-1-phosphate. In this configuration, fructose is readily cleaved by aldolase to form trioses that are the backbone for phospholipid and Triacylglycerol synthesis. Fructose also provides carbon atoms for synthesis  of  long-chain fatty acids, although in humans, the quantity of these carbon atoms is small. Thus, fructose facilitates the biochemical formation of triacylglycerols  more efficiently than does glucose.

Q.9-Why is it said that, ‘Fructose is not a direct energy source for muscles and the brain’?

Answer- These tissues rely on the hexokinase catalyzed phosphorylation of glucose for energy metabolism.  They do not take up fructose from the circulation since they lack both fructokinase and GLUT2. Fructose does increase hepatic fatty acid production and serum lipids and these can be utilized in muscle.  However, dyslipidemia is not a desirable situation. 

Q.10- Fructose has often been suggested as a treatment for hypoglycemia. There are several good reasons to discourage this.  Briefly explain the reasons.

Answer- Firstly the formation of glucose from Fructose is not an immediate process and

 

 

 

 

 

 

 

 

 

 

 

 

Figure 8- showing the fructose metabolism and energy expenditure

Secondly the conversion of fructose to glucose uses 2 ATPs. One ATP is spent at the first step for the formation of fructose-1-P and the second ATP is spent for the conversion of Glyceraldehyde to Glyceraldehyde-3-p (Figure 8).Normal hepatic activity alone utilizes all of the liver’s ATP-synthesizing capacity. There is no good reason to increase hepatic ATP utilization. Glucose metabolism on the other hand produces ATP instead of utilizing.

Moreover fructose is not a direct energy source for brain and muscle as these tissue lack fructokinase and fructose transporters while Glucose is a preferred fuel for brain and can be utilized in almost all the calls of the body. Hence Glucose (also called dextrose) instead of fructose can be given orally or intravenously depending upon the situation to treat hypoglycemia.

Q-11 -Parenteral feeding with solutions containing fructose can result in blood fructose concentrations that are several times higher than can be achieved with an oral load. What is the reason for this?

Answer- Since the rate of entry into the hepatocyte is dependent on the fructose gradient across the cell, intravenous loading results in  initial increased entry into the liver and increased formation of F1P. Since the rate of formation of F1P is much faster than its further metabolism,the accumulated fructose-1P, restricts  further flow of fructose in to the cells   with the resultant  increase in the  circulating  fructose load.

The toxic effects of F1P can also be exhibited  in the form of  hyperuricemia and hyperuricosuria by the mechanisms described separately.

 

 

 

 

 

 

 

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Q.1- Discuss the mechanism of digestion and absorption of fructose.

Answer-Fructose exists in foods as either a monosaccharide (free fructose) or as a unit of a disaccharide (sucrose). Free fructose is absorbed directly by the intestine; however, when fructose is consumed in the form of sucrose, digestion occurs entirely in the upper small intestine.As sucrose comes into contact with the membrane of the small intestine, the enzyme sucrase catalyzes the cleavage of sucrose to yield one glucose unit and one fructose unit. Fructose is absorbed in the small intestine, then enters the hepatic portal vein and is directed toward the liver.

Fructose absorption occurs on the mucosal membrane via facilitated transport involving GLUT5 transport proteins (Figure-1).Since the concentration of fructose is higher in the lumen, fructose is able to flow down a concentration gradient into the enterocytes, assisted by transport proteins. Fructose may be transported out of the enterocyte across the basolateral membrane by either GLUT2 or GLUT5, although the GLUT2 transporter has a greater capacity for transporting fructose and therefore the majority of fructose is transported out of the enterocyte through GLUT2. Fructose transfer activity increases with dietary fructose intake.The presence of fructose in the lumen causes increased mRNA transcription of GLUT5, leading to increased transport proteins.

 

 

 

 

 

 

 

 

Figure-1- showing the transport of fructose

Fructose Malabsorption- fructose malabsorption, formerly named “dietary fructose intolerance,” is a digestive disorder in which absorption of fructose is impaired by deficient fructose carriers (GLUT 5) in the small intestine’s enterocytes. This results in an increased concentration of fructose in the entire intestine. In the large intestine, fructose that hasn’t been adequately absorbed osmotically reduces the absorption of water and is metabolized by normal colonic bacteria to short chain fatty acids and the gases hydrogen, carbon dioxide and methane. This abnormal increase in hydrogen is detectable with the hydrogen breath test.

The physiological consequences of fructose malabsorption include increasing osmotic load, providing substrate for rapid bacterial fermentation and changing gastrointestinal motility. The presence of gases and organic acids in the large intestine causes gastrointestinal symptoms such as bloating, diarrhea, flatulence, and gastrointestinal pain.

 Restricting dietary intake of free fructose and/or fructans may provide symptomatic relief in a high proportion of patients. 

Q.2- How is fructose metabolized in the body? 

Answer- Much of the ingested fructose is metabolized by the liver, using the fructose 1-phosphate pathway (Figure-2). The first step is the phosphorylation of fructose to fructose 1-phosphate by fructokinase. Fructose 1-phosphate is then split into Glyceraldehyde and Dihydroxyacetone phosphate, an intermediate in glycolysis. This aldol cleavage is catalyzed by a specific fructose 1-phosphate aldolase. Glyceraldehyde is then phosphorylated to Glyceraldehyde 3-phosphate, a glycolytic intermediate, by triose kinase.

Figure-2 -showing the metabolism of fructose

Alternatively, fructose can be phosphorylated to fructose 6-phosphate by hexokinase. However, the affinity of hexokinase for glucose is 20 times as great as it is for fructose. In extrahepatic tissues, hexokinase catalyzes the phosphorylation of most hexose sugars, including fructose, but glucose inhibits the phosphorylation of fructose, since it is a better substrate for hexokinase. Little fructose 6- phosphate is formed in the liver because glucose is so much more abundant in this organ. Moreover, glucose, as the preferred fuel, is also trapped in the muscle by the hexokinase reaction. Because liver and muscle phosphorylate glucose rather than fructose, adipose tissue is exposed to more fructose than glucose. Hence, the formation of fructose 6- phosphate is not competitively inhibited to a biologically significant extent, and most of the fructose in adipose tissue is metabolized through fructose 6-phosphate. Fructose-6-p is phosphorylated by PFK-1 and is subsequently cleaved to form the two triose phosphates.

The two triose phosphates, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, may be degraded by glycolysis or may be substrates for aldolase and hence gluconeogenesis, which is the fate of much of the fructose metabolized in the liver.

Conversion of glucose to fructose- Fructose is found in seminal plasma. Glucose is reduced to Sorbitol in the presence of Aldose reductase(AR) and Sorbitol dehydrogenase(SD) is responsible for the conversion of Sorbitol into fructose (Figure-3). Fructokinase is present in the liver and seminal vesicles, hence fructose is metabolized by fructose-1-p pathway in these tissues.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3 showing the conversion of glucose to fructose in the liver and seminal vesicles by sorbitol pathway

Q.3- Why is it said that ingestion of large quantities of fructose or fructose containing syrups leads to dyslipidemia?

Or

Consumption of high-fructose corn syrup in beverages may play a role in the epidemic of obesity’, Justify the statement.

Answer- Diets high in sucrose or in high-fructose syrups (HFS) used in manufactured foods and beverages lead to large amounts of fructose (and glucose) entering the hepatic portal vein. Excessive fructose intake (>50 g/d) has been found to be one of the underlying etiologies of obesity, insulin resistance and metabolic syndrome. The mechanism responsible for the metabolic changes may be described as follows-

Synthesis of triglycerides - Carbons from dietary fructose are found in both the free fatty acid and glycerol moieties of plasma triglycerides. Fructose undergoes more rapid glycolysis in the liver than does glucose, because it bypasses the regulatory step catalyzed by phosphofructokinase (Figure-4). This allows fructose to flood the pathways in the liver. High fructose consumption can lead to excess pyruvate production, causing a buildup of Krebs cycle intermediates. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4- showing the cause of hypertriglyceridemia upon excessive fructose consumption

Accumulated citrate can be transported from the mitochondria into the cytosol of hepatocytes, converted to acetyl CoA by citrate lyase and directed toward fatty acid synthesis. Additionally, DHAP can be converted to glycerol 3-phosphate as previously mentioned, providing the glycerol backbone for the triglyceride molecule. Triglycerides are incorporated into very low density lipoproteins (VLDL), which are released from the liver destined toward peripheral tissues for storage in both fat and muscle cells. Excessive fatty acid and triglyceride levels are convincingly tied to development of the metabolic syndrome, hypertension, glucose intolerance and type 2 diabetes. Excessive fructose consumption is also believed to contribute to the development of non-alcoholic fatty liver disease.

In addition, unlike glucose, fructose does not stimulate insulin secretion or enhance leptin production. Because insulin and leptin act as key afferent signals in the regulation of food intake and body weight, this suggests that dietary fructose may contribute to increased energy intake and weight gain. Furthermore, calorically sweetened beverages may enhance caloric over consumption. Thus, the increase in consumption of HFCS (High fructose corn syrup) has a temporal relation to the epidemic of obesity, and the over consumption of HFCS in calorically sweetened beverages may play a role in the epidemic of obesity.

Q.4- What is the cause of hyperuricemia upon excessive fructose ingestion?

Answer- Fructose is mainly metabolized through Fructose-1-p pathway. Unlike phosphofructokinase, which is involved in glucose metabolism, fructokinase has no negative feedback system to prevent it from continuing to phosphorylate its substrate, i.e. Fructose to form fructose -1 phosphate, and as a consequence ATP can be depleted,(Figure 6 ) causing intracellular phosphate depletion, activation of AMP deaminase (Figure 5). AMP deaminase enzyme causes conversion of AMP to IMP (Inosine monophosphate). IMP, is subsequently converted to Hypoxanthine then to Xanthine and finally to Uric acid. Excessive uric acid generation leads to gout or renal stones.

 

 

 

 

 

 

 

Figure 5- AMP deaminase is inhibited by normal cellular concentrations of Pi. When these levels drop, the inhibition is released and AMP is converted to IMP and, ultimately, uric acid .

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-6 showing the cause of hyperuricemia on excessive fructose consumption

 

 

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Q. 5- Glucose 6-phosphate is metabolized by both the glycolytic pathway and the pentose phosphate pathway.

How is the processing of this important metabolite partitioned between these two metabolic routes?

Or

Write a balanced chemical reaction equation for the pentose pathway for each of the following modes: a) where ribose-5-phosphate synthesis is maximized b) where NADPH production is maximized, by conversion of sugar phosphate products to glucose-6-phosphate for repeated operation of the pathway c) where the fructose-6-phosphate and glyceraldehyde-3-phosphate generated in each passage through the pathway are catabolized via glycolysis and the citric acid cycle.

Answer- The Flow of Glucose 6-phosphate depends on the need for NADPH, Ribose 5-phosphate, and ATP. Glucose 6-phosphate can be metabolized in four different metabolic situations

Mode 1- Much more ribose 5-phosphate than NADPH is required For example, rapidly dividing cells need ribose 5-phosphate for the synthesis of nucleotide precursors of DNA. Most of the glucose 6-phosphate is converted into fructose 6-phosphate and glyceraldehyde 3-phosphate by the glycolytic pathway. Transaldolase and transketolase then convert two molecules of fructose 6-phosphate and one molecule of glyceraldehyde 3-phosphate into three molecules of ribose 5- phosphate by a reversal of the reactions as shown below

                       

 

 

 

 

 

 

The net reaction can be represented as follows-

 

 

 

Little or no ribose circulates in the bloodstream, so tissues have to synthesize the ribose they require for nucleotide and nucleic acid synthesis using the pentose phosphate pathway. It is not necessary to have a completely functioning pentose phosphate pathway for a tissue to synthesize ribose 5-phosphate. Muscle has only low activity of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, but, like most other tissues, it is capable of synthesizing ribose 5-phosphate by reversal of the nonoxidative phase of the pentose phosphate pathway utilizing fructose 6-phosphate.Thus Glycolytic and HMP pathway are linked together by Transaldolase and Transketolase. (Figure -1)

Mode 2- The needs for NADPH and ribose 5-phosphate are balanced. The predominant reaction under these conditions is the formation of two molecules of NADPH and one molecule of ribose 5-phosphate from one molecule of glucose 6- phosphate in the oxidative phase of the pentose phosphate pathway. The stoichiometry of mode 2 is-

 

 

 

Mode 3-  Much more NADPH than ribose 5-phosphate is required For example, adipose tissue requires a high level of NADPH for the synthesis of fatty acids, In this case, glucose 6-phosphate is completely oxidized to CO2.

Three groups of reactions are active in this situation. First, the oxidative phase of the pentose phosphate pathway forms two molecules of NADPH and one molecule of ribose 5-phosphate. Presuming that 6 molecules of glucose 6- phosphate enter the pathway, the net reaction would be-

 

 

 

 

Then, ribose 5-phosphate is converted into fructose 6-phosphate and glyceraldehyde 3-phosphate by transketolase and transaldolase. (See the reaction above for mode I)

 

 

 

 

 

Finally, glucose 6-phosphate is resynthesized from fructose 6-phosphate and glyceraldehyde 3-phosphate by the gluconeogenic pathway. The stoichiometry of these three sets of reactions is-

 

 

 

 

 

Glucose-6-P thus generated can re enter the pathway to produce more of NADPH.

Hence three pathways, HMP, Glycolysis and Gluconeogenesis interact to meet the excess needs of NADPH.

The sum of the mode 3 reactions is-

 

 

 

 

Thus, the equivalent of glucose 6-phosphate can be completely oxidized to CO2 with the concomitant generation of NADPH. (Figure 1)

Mode 4- Both NADPH and ATP are required. Alternatively, ribose 5-phosphate formed by the oxidative phase of the pentose phosphate pathway can be converted into pyruvate. Fructose 6-phosphate and glyceraldehyde 3-phosphate derived from ribose 5-phosphate enter the glycolytic pathway rather than reverting to glucose 6-phosphate. In this mode, ATP and NADPH are concomitantly generated, and five of the six carbons of glucose 6-phosphate emerge in pyruvate.

 

 

 

 

 

Pyruvate formed by these reactions can be oxidized to generate more ATP or it can be used as a building block in a variety of biosynthesis.(Figure -1)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure -1- showing the four modes of pentose phosphate pathway

Q.6 – Discuss the metabolic significance of HMP pathway.

Answer-The pentose phosphate pathway is primarily an anabolic pathway that utilizes 6 carbons of glucose to generate 5 carbon sugars and reducing equivalents. However, this pathway does oxidize glucose and under certain conditions can completely oxidize glucose to CO2 and water. The primary functions of this pathway are:

1. To generate reducing equivalents, in the form of NADPH, for reductive biosynthesis reactions within cells. 

The examples of reactions requiring NADPH are as follows-

i) De novo fatty acid synthesis

ii) Synthesis of cholesterol

iii) Synthesis of steroids

iv) Synthesis of sphingolipids

v) Synthesis of neurotransmitters

vi) Microsomal desaturation of fatty acids

vii) Conversion of phenyl Alanine to tyrosine

viii) Drug detoxification

ix) Reduction of glutathione

x) Reduction of folate

xi) Reduction of Met Hb to normal Hb

xii) The conversion of ribonucleotides to deoxy ribonucleotides (through the action of ribonucleotide reductase) requires NADPH as the electron source; therefore, any rapidly proliferating cell needs large quantities of NADPH.

xiii) Macrophageal functions

2. To provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides, nucleic acids, ATP and coenzymes.

3. Although not a significant function of the PPP, it can operate to metabolize dietary pentose sugars derived from the digestion of nucleic acids as well as to rearrange the carbon skeletons of dietary pentoses into glycolytic/gluconeogenic intermediates. Glyceraldehyde-3-P and fructose-6-P formed from 5‐C sugar phosphates may enter Glycolysis for ATP synthesis. The Pentose Phosphate Pathway thus serves as an entry into Glycolysis for both 5‐carbon & 6‐carbon sugars.

4. CO2 produced from this pathway can be utilized for CO2 fixation reactions.

Q. 7- What are the predominant pathways of Glucose utilization in Erythrocytes? Give the significance of each. 

Answer- The predominant pathways of carbohydrate metabolism in the red blood cells (RBC) are glycolysis, the PPP and 2,3-bisphosphoglycerate pathway. Glycolysis provides ATP for membrane ion pumps and NADH for re-oxidation of methemoglobin. 2,3-bisphosphoglycerate is required for unloading of O2 to the peripheral tissues.

The PPP supplies the RBC with NADPH to maintain the reduced state of Glutathione (Figure-2)

 

 

 

 

 

 

 

 

 

 

 

Figure-2 showing the reduction of glutathione through NADPH formed in HMP pathway

The inability to maintain reduced glutathione in RBCs leads to increased accumulation of peroxides, predominantly H2O2, that in turn results in a weakening of the cell membrane and concomitant hemolysis. Accumulation of H2O2 also leads to increased rates of oxidation of hemoglobin to methemoglobin that also weakens the cell wall. Glutathione removes peroxides via the action of glutathione peroxidase. The PPP in erythrocytes is essentially the only pathway for these cells to produce NADPH. Any defect in the production of NADPH could, therefore, have profound effects on erythrocyte survival.

Deficiency in the level of activity of glucose-6-phosphate dehydrogenase (G6PDH) is the basis of favism, primaquine (an anti-malarial drug) sensitivity and some other drug-sensitive hemolytic anemias, anemia and jaundice in the newborn, and chronic nonspherocytic hemolytic anemia. In addition, G6PDH deficiencies are associated with resistance to the malarial parasite, Plasmodium falciparum, among individuals of Mediterranean and African descent.The basis for this resistance is the weakening of the red cell membrane (the erythrocyte is the host cell for the parasite) such that it cannot sustain the parasitic life cycle long enough for productive growth.

Q.8- Is it correct to say that excessive carbohydrate ingestion leads to obesity?

Answer- Yes, it is true. Excessive carbohydrate ingestion promotes triglyceride synthesis through following mechanisms-

1) Glycolysis yields pyruvate and hence Acetyl coA which is a precursor for fatty acid biosynthesis.

2) Glycolysis provides glycerol-3-p through dihydroxyacetone phosphate

3) HMP pathway provides NADPH which can be used for reductive biosynthesis.

By all these mechanisms, fatty acids are synthesized and esterified with glycerol to produce triglycerides. The adipose mass increases and the person gets obese.

Q.9- What is the reason that individuals with reduced ability to produce NADPH are at increased risk for specific recurrent infections?

Answer- The highest levels of PPP enzymes (in particular glucose 6-phosphate dehydrogenase) are found in neutrophils and macrophages. These leukocytes are the phagocytic cells of the immune system and they utilize NADPH to generate superoxide radicals from molecular oxygen in a reaction catalyzed by NADPH oxidase. Superoxide anion, in turn, serves to generate other reactive oxygen species (ROS) the kill the phagocytized microorganisms. Following exposure to bacteria and other foreign substances there is a dramatic increase in O2 consumption by phagocytes. This phenomenon is referred to as the oxygen burst.

Because of the need for NADPH in phagocytic cells, by the NADPH oxidase system, any defect in enzymes in this process can result in impaired killing of infectious organisms. Chronic granulomatous disease, CGD is a syndrome that results in individuals harboring defects in the NADPH oxidase system. There are several forms of CGD involving defects in various components of the NADPH oxidase system. Individuals with CGD are at increased risk for specific recurrent infections. The most common are pneumonia, abscesses of the skin, tissues, and organs, suppurative arthritis (invasion of the joints by infectious agent leading to generation of pus), and Osteomyelitis (infection of the bone). The majority of patients with CGD harbor mutations in an X-chromosome gene that encodes a component of the NADPH oxidase system. Given the role of NADPH in the process of phagocytic killing it is clear that individuals with reduced ability to produce NADPH (such as those with G6PDH deficiencies) may also manifest with symptoms of CGD.

Q 10.-  What two products of the linear portion of the Pentose Phosphate Pathway have essential roles in anabolic metabolism? What are these roles?

Answer- The linear portion of the pathway carries out oxidation and decarboxylation of glucose-6-phosphate, producing the 5-C sugar ribulose-5-phosphate. NADP+ serves as electron acceptor. Ribulose-5-phosphate is isomerized to Ribose-5-phosphate, that  can be used for the synthesis of nucleotides and coenzymes. NADPH the product of linear portion of the pathway is used for reductive biosynthesis like fatty acid synthesis, cholesterol or steroid biosynthesis etc. The reversible or non oxidative phase of HMP pathway is also called cyclic portion of HMP pathway.

 Q.11-  If a patient has glucose-6-phosphate dehydrogenase deficiency, why are red blood cells lysed while other cells of the body remain intact? What is the biochemical basis for hemolysis? Why doesn’t this disease show up earlier in life? Give a brief account of the drug induced hemolytic anemia in G6 PD deficiency.

Or

A 34- year-old African – American man was seen with fever and shortness of breath. Shortly afterwards he developed pancreatitis and was treated with an antibiotic, clindamycin and primaquine. After four days in to this therapy the onset of hematuria was noted. The patient’s Hb fell from 11.0g/dl to 7.4g/dl, his total bilirubin increased from 1.2 mg/dl to 4.3 mg/dl.

What is the probable diagnosis?

What is the relationship of Primaquin and hemolytic anemia?

Answer- The patient is most probably suffering from Glucose -6- phosphate dehydrogenase deficiency. The hemolysis is primaquine induced which is an oxidant drug. The rise in bilirubin is due to hemolytic jaundice

Reactive oxygen species (ROS) generated in oxidative metabolism inflict damage on all classes of macromolecules and can ultimately lead to cell death. Indeed, ROS are implicated in a number of human diseases. Reduced glutathione (GSH), a tripeptide with a free sulfhydryl group, is required to combat oxidative stress and maintain the normal reduced state in the cell. Oxidized glutathione (GSSG) is reduced by NADPH generated by glucose 6-phosphate dehydrogenase in the pentose phosphate pathway (Figure-2). Indeed, cells with reduced levels of glucose 6-phosphate dehydrogenase are especially sensitive to oxidative stress. This stress is most acute in red blood cells because, lacking mitochondria; they have no alternative means of generating reducing power.

 

 

 

 

 

 

 

 

 

 

Figure 2- showing the formation of NADPH Through oxidative phase of HMP pathway

 

Glucose- 6- phosphate dehydrogenase deficiency

Glucose-6-phosphatase dehydrogenase (G6PD) deficiency is the most common disease-producing enzymopathy in humans. Inherited as an X-linked disorder, glucose-6-phosphatase dehydrogenase (G6PD) deficiency affects 400 million people worldwide.

G6PD deficiency is a prime example of a hemolytic anemia due to interaction between an intracorpuscular and an extracorpuscular cause, because in the majority of cases hemolysis is triggered by an exogenous agent. People deficient in glucose-6-phosphatase dehydrogenase (G6PD) are not prescribed oxidative drugs, because their red blood cells undergo rapid hemolysis under this stress. Although in G6PD-deficient subjects there is a decrease in G6PD activity in most tissues, this is less marked than in red cells, and it does not seem to produce symptoms.

Clinical Manifestations

1) The vast majority of people with G6PD deficiency remain clinically asymptomatic throughout their lifetime.

2) However, there is an increased risk of developing neonatal jaundice (NNJ) and a risk of developing acute HA when challenged by a number of oxidative agents.

3) The onset can be extremely abrupt, especially with favism in children. The anemia is moderate to extremely severe, usually normocytic normochromic, and due partly to intravascular hemolysis; hence, it is associated with haemogobinemia and  hemoglobinuria,

Precipitating factors

Acute HA can develop as a result of three types of triggers: (1) fava beans, (2) infections, and (3) drugs like- Antimalarials, antibiotics, antipyretics/ analgesics, sulfonamides etc

The presence of pamaquine, a purine glycoside of fava beans, or other nonenzymatic oxidative agents leads to the generation of peroxides, reactive oxygen species that can damage membranes as well as other biomolecules. Peroxides are normally eliminated by glutathione peroxidase with the use of glutathione as a reducing agent.

 

 

 

 

Moreover, in the absence of the enzyme, the hemoglobin sulfhydryl groups can no longer be maintained in the reduced form and hemoglobin molecules then cross-link with one another to form aggregates called Heinz bodies on cell membranes. A membrane damaged by the Heinz bodies and reactive oxygen species become deformed and the cell is likely to undergo lysis. In the absence of oxidative stress, however, the deficiency is quite benign.

Identification and discontinuation of the precipitating agent is critical in cases of glucose-6-phosphatase dehydrogenase (G6PD) deficiency. Affected individuals are treated with oxygen and bed rest, which may afford symptomatic relief. Prevention of drug-induced hemolysis is possible in most cases by choosing  alternative drugs.

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Q.1- What is HMP pathway? Explain in detail the steps of this pathway.

Answer- The pentose phosphate pathway (also called Phospho gluconate pathway or hexose monophosphate shunt [HMP shunt]) is an alternative route for the metabolism of glucose. It does not lead to formation of ATP but has two major functions: (1) The formation of NADPH for synthesis of fatty acids and steroids, and (2) the synthesis of ribose for nucleotide and nucleic acid formation.

Overview of HMP pathway

The pentose phosphate pathway meets the need of all organisms for a source of NADPH to use in reductive biosynthesis .This pathway consists of two phases: the oxidative generation of NADPH and the nonoxidative interconversion of sugars (Figure 1). In the oxidative phase, NADPH is generated when glucose 6-phosphate is oxidized to ribose 5-phosphate. This five-carbon sugar and its derivatives are components of RNA and DNA, as well as ATP, NADH, FAD, and coenzyme A.

In the nonoxidative phase, the pathway catalyzes the interconversion of three-, four-, five-, six-, and seven-carbon sugars in a series of nonoxidative reactions that can result in the synthesis of five-carbon sugars for nucleotide biosynthesis or the conversion of excess five-carbon sugars into intermediates of the glycolytic pathway. All these reactions take place in the cytosol.

Figure 1- showing an overview of HMP pathway- GAP (Glyceraldehyde-3-phosphate)

The pentose phosphate pathway is a more complex pathway than glycolysis (Figure 1). Six molecules of glucose 6-phosphate give rise to six molecules of CO2 and six five-carbon sugars, these are rearranged to regenerate 4 molecules of glucose 6-phosphate and 2 molecule of the glycolytic intermediate, glyceraldehyde 3-phosphate. Since two molecules of glyceraldehyde 3-phosphate can regenerate glucose 6-phosphate, hence 5 molecules glucose 6-phosphate are regenerated. The generation of 6 molecules of CO2 in  the pathway can account for the complete oxidation of  one molecule of glucose. 

Reactions of HMP pathway

The sequence of reactions of the pathway may be divided into two phases: an oxidative nonreversible phase and a nonoxidative reversible phase.

1) Oxidative Phase-Unlike glycolysis, oxidation is achieved by dehydrogenation using NADP+, not NAD+, as the hydrogen acceptor. The oxidative phase of the pentose phosphate pathway starts with the dehydrogenation of glucose 6-phosphate at carbon 1, a reaction catalyzed by glucose 6-phosphate dehydrogenase (Figure 2). This enzyme is highly specific for NADP+; the Km for NAD+ is about a thousand times as great as that for NADP+. The product is 6-phosphoglucono-δ-lactone, which is an intramolecular ester between the C-1 carboxyl group and the C-5 hydroxyl group. The next step is the hydrolysis of 6-phosphoglucono- δ -lactone,  by a specific lactonase(Hydrolase) to give 6-phosphogluconate. This six-carbon sugar is then oxidatively decarboxylated by 6-phosphogluconate dehydrogenase to yield Ribulose 5-phosphate. NADP+ is again the electron acceptor. The final step in the synthesis of ribose 5-phosphate is the isomerization of Ribulose 5-phosphate by phosphopentose Isomerase (see Figure)

Figure 2- showing the reactions of oxidative phase of HMP pathway

The Nonoxidative Phase

Ribulose 5-phosphate is the substrate for two enzymes. Ribulose 5-phosphate 3-epimerase(Phosphopentose epimerase) alters the configuration about carbon 3, forming the epimer Xylulose 5-phosphate, also a ketopentose. (Figure 3)

Ribose 5-phosphate keto Isomerase (Phosphopentose isomerase) converts Ribulose 5-phosphate to the corresponding aldopentose, ribose 5-phosphate, which is the precursor of the ribose required for nucleotide and nucleic acid synthesis.(Figure 3)

Figure 3- showing interconversion of pentoses

Transketolase transfers the two-carbon unit comprising carbons 1 and 2 of a ketose onto the aldehyde carbon of an aldose sugar. It therefore effects the conversion of a ketose sugar into an aldose with two carbons less and an aldose sugar into a ketose with two carbons more. The reaction requires Mg2+ and thiamin diphosphate (vitamin B1) as coenzyme. The two-carbon moiety transferred is probably glycoaldehyde bound to thiamin diphosphate. Thus, transketolase catalyzes the transfer of the two-carbon unit from Xylulose 5-phosphate to ribose 5-phosphate, producing the seven-carbon ketose sedoheptulose 7-phosphate and the aldose glyceraldehyde 3-phosphate. These two products then undergo transaldolation.(Figure 4)

Figure 4- showing the reaction catalyzed by transketolase

Transaldolase catalyzes the transfer of a three-carbon Dihydroxyacetone moiety (carbons 1–3) from the ketose sedoheptulose 7-phosphate onto the aldose glyceraldehyde 3-phosphate to form the ketose fructose 6-phosphate and the four-carbon aldose erythrose 4-phosphate. (Figure-5)

Figure 5- showing the reaction catalyzed by transaldolase

In a further reaction catalyzed by transketolase, Xylulose 5-phosphate serves as a donor of glycoaldehyde. In this case erythrose 4-phosphate is the acceptor, and the products of the reaction are fructose 6-phosphate and glyceraldehyde 3-phosphate. (Figure 6)

Figure 6- showing the rearrangement of sugars to form glycolytic intermediated catalyzed by transketolase

In order to oxidize glucose completely to CO2 via the pentose phosphate pathway, there must be enzymes present in the tissue to convert glyceraldehyde 3-phosphate to glucose 6-phosphate. This involves reversal of glycolysis and the gluconeogenic enzyme fructose 1,6-bisphosphatase. In tissues that lack this enzyme, glyceraldehyde 3-phosphate follows the normal pathway of glycolysis to pyruvate.

The sum of these reactions is

Xylulose 5-phosphate can be formed from ribose 5-phosphate by the sequential action of phosphopentose isomerase and phosphopentose Epimerase, and so the net reaction starting from ribose 5-phosphate is

Thus, excess ribose 5-phosphate formed by the pentose phosphate pathway can be completely converted into glycolytic intermediates. Moreover, any ribose ingested in the diet can be processed into glycolytic intermediates by this pathway.

It is evident that the carbon skeletons of sugars can be extensively rearranged to meet physiologic needs.

Q.2- Enlist the important differences between Glycolysis and HMP pathway.

 Answer-

Characteristics Glycolysis HMP pathway
Occurrence All cells of the body Active in liver, adipose tissue, adrenal cortex, thyroid, erythrocytes, testis, and lactating mammary glands.
Glucose Oxidation(Coenzyme) Oxidation is achieved by dehydrogenation using NAD+ as the hydrogen acceptor. Oxidation is achieved by dehydrogenation using NADP+ as the hydrogen acceptor.
CO2 production CO2 is not produced CO2 is produced
Pentose production Pentoses are not produced Pentoses are produced
Intermediates Can be in the bisphosphate form- such as Fr 1,6 bisphosphate , 1,3 bisphosphoglycerate or 2,3 bisphosphoglycerate etc. Never in bisphosphate form. Always in mono phosphate form that is why called Hexose mono phosphate pathway.
Energy ATP is utilized as well as produced. ATP is a major product of glycolysis ATP is neither utilized nor produced.Glycolytic intermediates may enter glycolytic pathway to produce energy
 Biological Significance Energy production both in aerobic and anaerobic conditions NADPH is required for reductive biosynthesis and pentoses are required forsynthesis of coenzymes and nucleotides.
Clinical Significance Hemolytic anemia in Pyruvate kinase and Hexokinase deficiency Hemolytic anemia in Glucose-6-P dehydrogenase deficiency

 

Q.3- The pentose phosphate pathway is active in liver, adipose tissue, adrenal cortex, thyroid, erythrocytes, testis, and lactating mammary gland. Why is the pathway less active in non-lactating mammary gland and skeletal muscle? 

Answer- HMP pathway is highly active in rapidly dividing cells and in tissues where there is a great requirement of NADPH. The reactions of fatty acid biosynthesis and steroid biosynthesis utilize large amounts of NADPH. As a consequence, cells of the liver, adipose tissue, adrenal cortex, testis and lactating mammary gland have high levels of the PPP enzymes. In fact 30% of the oxidation of glucose in the liver occurs via the PPP. Additionally, erythrocytes utilize the reactions of the PPP to generate large amounts of NADPH used in the reduction of glutathione

In liver NADPH is required for the synthesis of fatty acids, sterols, cholesterol and for the activity of Glutamate dehydrogenase,

In adipose tissue and in lactating mammary glands, NADPH is required for fatty acids synthesis.

It is required for the synthesis of steroid hormones in adrenal cortex

Similarly it is required for the synthesis of hormones in testes, ovaries and in thyroid gland.

In lens and in Erythrocytes NADPH is required for reduction of Glutathione which is an essential component of Glutathione peroxidase, meant for maintaining the integrity of these tissues.

In skeletal, muscle, this pathway is less active. Muscle tissue contains very small amount of dehydrogenases but skeletal muscle is capable of synthesizing ribose. Probably, this is accomplished by reversal of non oxidative phase of shunt pathway utilizing fructose-6-P and Glyceraldhyde-3-P under the activities of Transketolase and Transaldolase enzymes

Q.4- Discuss the regulation of HMP pathway.  Under what conditions is this pathway stimulated or inhibited?

Answer- Glucose-6-phosphate Dehydrogenase is the committed step of the Pentose Phosphate Pathway.  Following factors affect the activity of this enzyme and thus influence the rate of this pathway-

 I) Availability of Substrate- This enzyme is regulated by availability of the substrate NADP+. As NADPH is utilized in reductive synthetic pathways, the increasing concentration of NADP+ stimulates the Pentose Phosphate Pathway, to replenish NADPH.  The inhibitory effect of low levels of NADP+ is exacerbated by the fact that NADPH competes with NADP+ in binding to the enzyme. The low ratio of NADP+ to NADPH stimulates the enzyme while the high ratio inhibits the enzyme. The marked effect of the NADP+ level on the rate of the oxidative phase ensures that NADPH generation is tightly coupled to its utilization in reductive biosyntheses. This explains the higher rate of activity of HMP pathway in tissues involved in reductive biosynthesis.

2) Induction and repression- The synthesis of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase may also be induced by insulin in the fed state, when lipogenesis increases.  Excessive carbohydrate ingestion thereby leads to more NADPH generation.

The synthesis of these enzymes is decreased in the fasting state.

The non oxidative phase of HMP pathway accomplishes conversion of the 5-C ribulose-5-phosphate to the 5-C product ribose-5-phosphate, or to the 3-C glyceraldehyde-3-phosphate and the 6-C fructose-6-phosphate. This phase is regulated by the flow of substrates.

 

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Q.1-What is the cause of Alcohol induced hypoglycemia?

Answer- Alcohol-related hypoglycemia is due to hepatic glycogen depletion combined with alcohol-mediated inhibition of gluconeogenesis. It is most common in malnourished alcohol abusers, but can occur in anyone who is unable to ingest food after an acute alcoholic episode followed by gastritis and vomiting.

The primary pathway for alcohol metabolism involves alcohol dehydrogenase (ADH), a cytosolic enzyme that catalyzes the conversion of alcohol to acetaldehyde (Figure-1). This enzyme is located mainly in the liver, but small amounts are found in other organs such as the brain and stomach.

During conversion of ethanol by ADH to acetaldehyde, hydrogen ion is transferred from alcohol to the cofactor nicotinamide adenine dinucleotide (NAD+) to form NADH.

Much of the acetaldehyde formed from alcohol is oxidized in the liver in a reaction catalyzed by mitochondrial NAD-dependent aldehyde dehydrogenase (AcDH). The product of this reaction is acetate (Figure-1), which can be further metabolized to CO2 and water, or used to form acetyl-CoA. As a net result, alcohol oxidation generates an excess of reducing equivalents in the liver, chiefly as NADH. The excess NADH production appears to contribute to the metabolic disorders that accompany chronic alcoholism.

The NADH produced in the cytosol by ADH must be reduced back to NAD+. The reduction in NAD+ Impairs the flux of glucose through glycolysis at the glyceraldehyde-3-phosphate dehydrogenase reaction, thereby limiting energy production. Additionally, there is an increased rate of hepatic lactate production due to the effect of increased NADH on the direction of the hepatic lactate dehydrogenase (LDH) reaction. This reversal of the LDH reaction in hepatocytes diverts pyruvate from gluconeogenesis leading to a reduction in the capacity of the liver to deliver glucose to the blood.

In addition to the negative effects of the altered NADH/NAD+ ratio on hepatic gluconeogenesis, fatty acid oxidation is also reduced as this process requires NAD+ as a cofactor. In fact the opposite is true, fatty acid synthesis is increased and there is an increase in triacylglyceride production by the liver. In the mitochondria, the production of acetate from acetaldehyde leads to increased levels of acetyl-CoA. Since the increased generation of NADH also reduces the activity of the TCA cycle, the acetyl-CoA is diverted to fatty acid synthesis. The reduction in cytosolic NAD+ leads to reduced activity of glycerol-3-phosphate dehydrogenase (in the glycerol 3-phosphate to DHAP direction) resulting in increased levels of glycerol 3-phosphate which is the backbone for the synthesis of the triacylglycerides. Both of these two events lead to fatty acid deposition in the liver, leading to fatty liver syndrome.

  

 

Figure-1- showing metabolism of alcohol and conversion of pyruvate to lactate

Q.-2 Premature and low-birth-weight babies are more susceptible to hypoglycemia, what could be the possible cause for this?

Answer- Premature and low-birth-weight babies are more susceptible to hypoglycemia, since they have little adipose tissue to provide alternative fuels such as free fatty acids or ketone bodies during the transition from fetal dependency to the free-living state. The enzymes of gluconeogenesis may not be completely functional at this time, and gluconeogenesis is any way dependent on a supply of free fatty acids for energy. Little glycerol, which would normally be released from adipose tissue, is available for gluconeogenesis, but that is not sufficient to fulfill the energy needs. Small for date babies have inadequate glycogen stores as well, so at the time of need there is diminished outpouring of glucose. The situation worsens further due to prematurity since the glycogen stores are laid in the last months of pregnancy. Hence a premature baby has diminished stores and frequently undergoes hypoglycemia.

Q.3- What is the role played by kidneys in gluconeogenesis?

Answer- Although the liver has the critical role of maintaining blood glucose homeostasis and therefore, is the major site of gluconeogenesis, the kidney plays an important role. During periods of severe hypoglycemia that occur under conditions of hepatic failure, the kidney can provide glucose to the blood via renal gluconeogenesis. In the renal cortex, glutamine is the preferred substance for gluconeogenesis.

Glutamine is produced in high amounts by skeletal muscle during periods of fasting as a means to export the waste nitrogen resulting from amino acid catabolism. Through the actions of transaminases, a mole of waste ammonia is transferred to α-ketoglutarate via the glutamate dehydrogenase catalyzed reaction yielding glutamate. Glutamate is then a substrate for glutamine synthetase, which incorporates another mole of waste ammonia generating glutamine .The glutamine is then transported to the kidneys where the reverse reactions occur liberating the ammonia and producing α-ketoglutarate which can enter the TCA cycle and the carbon atoms diverted to gluconeogenesis via oxaloacetate. This process serves two important functions. The ammonia (NH3) that is liberated spontaneously ionizes to ammonium ion (NH4+) and is excreted in the urine effectively buffering the acids in the urine. In addition, the glucose that is produced via gluconeogenesis can provide the brain with critically needed energy.

 Q.4-   What is the biochemical basis for-

 a) Hypoglycemia in- Babies of diabetic mothers

 b) Maternal hypoglycemia during pregnancy

Answer- a) Babies of diabetic mothers- The growing fetus of a diabetic mother is exposed to maternal hyperglycemia which leads to hyperplasia of pancreatic islet cells. After delivery the baby fails to suppress the excessive insulin secretions and develops hypoglycemia.

b) Maternal or fetal hypoglycemia may also be observed during pregnancy, fetal glucose consumption increases and there is a risk of maternal and possibly fetal hypoglycemia, particularly if there are long intervals between meals or at night.

Q.5- What is the biochemical basis for Hypoglycemia in conditions of impaired fatty acid oxidation ?

Answer- Impaired fatty acid oxidation can be due to

1) Deficiency or inactivation of any of the enzyme of fatty acid oxidation pathway.

2) Carnitine deficiency (Carnitine is a transporter for transportation of fatty acids from the cytoplasm to mitochondria where the actual oxidation takes place).

3) Deficiency of coenzyme, as in chronic alcoholism

Impaired fatty acid oxidation results in hypoglycemia due to three main reasons-

a) There is an imbalance between demand and supply of fuels. There is no fatty acid oxidation, the requirement for Glucose increases

b) Acetyl Co A, the end product of fatty acid oxidation acts as a positive modifier for pyruvate carboxylase enzyme (Figure-2). In  conditions of impaired fatty acid oxidation, there is less activity of pyruvate carboxylase, less oxaloacetate  and hence less glucose production.

c) The energy evolved from fatty acid oxidation is used for glucose production in conditions of fasting or starvation, but in conditions of impaired fatty acid oxidation, non availability of sufficient energy causes inhibition of the pathway resulting in hypoglycemia.

 

Figure-2 -Acetyl Co A acts a  negative modifier for Pyruvate dehydrogenase complex (PDH) while a positive modifier for Pyruvate carboxylase. In conditions of excess production of  Acetyl co A  (as during fasting,starvation or diabetes mellitus, there is excessive fatty acid oxidation) PDH complex is inhibited, while pyruvate carboxylase is stimulated to provide more glucose.

Q.6-Why do nutritionists recommend  very low carbohydrate diets  to reduce body weight, What is the biochemical basis ?

Answer- Very low carbohydrate diets, providing only 20 g per day of carbohydrate or less (compared with a desirable intake of 100–120 g/day), but permitting unlimited consumption of fat and protein, have been promoted as an effective regime for weight loss, although such diets are counter to all advice on a prudent diet for health. Since there is a continual demand for glucose, there will be a considerable amount of gluconeogenesis from amino acids; the associated high ATP cost must then be met by oxidation of fatty acids. Endogenous adipose stores are depleted with the resultant weight loss.

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Q.1-Comment on the statement – ‘Glycolysis and Gluconeogenesis are reciprocally regulated’

Or

Discuss the regulation of Gluconeogenesis. 

Answer- Gluconeogenesis and glycolysis are coordinated so that within a cell one pathway is relatively inactive while the other is highly active. The amounts and activities of the distinctive enzymes of each pathway are controlled so that both pathways are not highly active at the same time.

Changes in the availability of substrates are responsible for most changes in metabolism either directly or indirectly acting via changes in hormone secretion. Three mechanisms are responsible for regulating the activity of enzymes (1) changes in the rate of enzyme synthesis, (Induction/Repression) (2) covalent modification by reversible phosphorylation, and (3) allosteric effects.

1) Induction & Repression of Key Enzymes

The amounts and the activities of essential enzymes are regulated by hormones. The enzymes involved catalyze nonequilibrium (physiologically irreversible) reactions. Hormones affect gene expression primarily by changing the rate of transcription, as well as by regulating the degradation of mRNA.

Insulin, which rises subsequent to eating, stimulates the expression of phosphofructokinase-1, pyruvate kinase, and the bifunctional enzyme that makes and degrades Fructose-2,6-BP (PFK-2 and Fructose 2,6 bisphosphatase). PFK2 is mainly affected by Insulin to increase the concentration of Fructose-2,6 bisphosphate.

Glucagon, which rises during starvation, inhibits the expression of these enzymes and stimulates instead the production of two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and fructose 1,6-bisphosphatase. Transcriptional control in eukaryotes is much slower than allosteric control; it takes hours or days in contrast with seconds to minutes.

2) Covalent Modification by Reversible Phosphorylation

It is a rapid process. Glucagon and epinephrine, hormones that are responsive to a decrease in blood glucose, inhibit glycolysis and stimulate gluconeogenesis in the liver by increasing the concentration of cAMP. This in turn activates cAMP-dependent protein kinase, leading to the phosphorylation and inactivation of pyruvate kinase. They also affect the concentration of fructose 2,6-bisphosphate by activating Fructose 2,6 bisphoaphatase and therefore glycolysis and gluconeogenesis are appropriately regulated. Low concentration of fructose 2,6  bisphosphate reduces the activity of PFK-I and thereby reduces the rate of glycolysis, while at the same time since fructose 2,6  bisphosphate  inhibits the activity of Fr 1,6 bis phosphatase,its low concentration overcomes the inhibition and gluconeogensis is stimulated.

3) Allosteric Modification

It is an instantaneous process.The role of various allosteric modifiers can be explained as follows-

a) Role of Acetyl co A

In gluconeogenesis, pyruvate carboxylase, which catalyzes the synthesis of oxaloacetate from pyruvate, requires acetyl-CoA as an allosteric activator. The addition of acetyl-CoA results in a change in the tertiary structure of the protein, lowering the Km for bicarbonate (CO2 is added in the form of bicarbonate).

The activation of pyruvate carboxylase and the reciprocal inhibition of pyruvate dehydrogenase by acetyl-CoA derived from the oxidation of fatty acids explain the action of fatty acid oxidation in sparing the oxidation of pyruvate and in stimulating gluconeogenesis. The reciprocal relationship between these two enzymes alters the metabolic fate of pyruvate as the tissue changes from carbohydrate oxidation (glycolysis) to gluconeogenesis during the transition from the fed to fasting state.

b)  Role of ATP and AMP

The interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate is stringently controlled (Figure-1). Phosphofructokinase (phosphofructokinase-1) occupies a key position in regulating glycolysis and is also subject to feedback control.  AMP stimulates phosphofructokinase, whereas ATP and citrate inhibit it. Fructose 1,6- bisphosphatase, on the other hand, is inhibited by AMP and activated by citrate. A high level of AMP indicates that the energy charge is low and signals the need for ATP generation. Conversely, high levels of ATP and citrate indicate that the energy charge is high and that biosynthetic intermediates are abundant. Under these conditions, glycolysis is nearly switched off and gluconeogenesis is promoted.

The interconversion of phosphoenolpyruvate and pyruvate also is precisely regulated. Pyruvate kinase is controlled by allosteric effectors and by phosphorylation. High levels of ATP and alanine, which signal that the energy charge is high and that building blocks are abundant, inhibit the enzyme in liver. Likewise, ADP inhibits phosphoenolpyruvate carboxykinase. Hence, gluconeogenesis is favored when the cell is rich in biosynthetic precursors and ATP.

c) Role of Fructose 2,6-Bisphosphate

The most potent positive allosteric activator of phosphofructokinase-1 and inhibitor of fructose 1,6-bisphosphatase in liver is fructose 2,6-bisphosphate.  It is a positive modifier of PFK-1, and a negative modifier of Fr, 1,6 bisphoaphatase.It relieves inhibition of phosphofructokinase-1 by ATP and increases the affinity for fructose 6-phosphate. It inhibits fructose 1,6-bisphosphatase by increasing the Km for fructose 1,6-bisphosphate.

Its concentration is under both substrate (allosteric) and hormonal control (covalent modification) (Figure-1). Fructose 2,6-bisphosphate is formed by phosphorylation of fructose 6-phosphate by phosphofructokinase-2. The same enzyme protein is also responsible for its breakdown, since it has fructose 2,6-bisphosphatase activity. This bifunctional enzyme is under the allosteric control of fructose 6-phosphate, which stimulates the kinase and inhibits the phosphatase.

Hence, when there is an abundant supply of glucose, the concentration of fructose 2,6-bisphosphate increases, stimulating glycolysis by activating phosphofructokinase-1 and inhibiting fructose 1,6-bisphosphatase. In the fasting state, glucagon stimulates the production of cAMP, activating cAMP-dependent protein kinase, which in turn inactivates phosphofructokinase-2 and activates fructose 2,6-bisphosphatase by phosphorylation. Hence, gluconeogenesis is stimulated by a decrease in the concentration of fructose 2,6-bisphosphate, which inactivates phosphofructokinase-1 and relieves the inhibition of fructose 1,6-bisphosphatase.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-1- Reciprocal Regulation of Gluconeogenesis and Glycolysis in the Liver,  The level of fructose 2,6-bisphosphate is high in the fed state and low in starvation. Another important control is the inhibition of pyruvate kinase by phosphorylation during starvation.

Q.2-What do you know about substrate cycle or futile cycle? 

Answer- A pair of reactions such as the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate and its hydrolysis back to fructose 6-phosphate is called a substrate cycle. Both reactions are not simultaneously fully active in most cells, because of reciprocal allosteric controls. However, the results of isotope-labeling studies have shown that some fructose 6-phosphate is phosphorylated to fructose 1,6-bisphosphate in gluconeogenesis. There also is a limited degree of cycling in other pairs of opposed irreversible reactions. This cycling was regarded as an imperfection in metabolic control, and so substrate cycles have sometimes been called futile cycles

In muscle, both phosphofructokinase and fructose 1,6-bisphosphatase have some activity at all times, so that there is indeed some measure of (wasteful) substrate cycling. This permits the very rapid increase in the rate of glycolysis necessary for muscle contraction. At rest, the rate of phosphofructokinase activity is some tenfold higher than that of fructose 1,6-bisphosphatase; in anticipation of muscle contraction, the activities of both enzymes increase, fructose 1,6-bisphosphatase ten times more than phosphofructokinase, maintaining the same net rate of glycolysis. At the start of muscle contraction, the activity of phosphofructokinase increases further, and that of fructose 1,6-bisphosphatase falls, so increasing the net rate of glycolysis (and hence ATP formation) as much as a thousand fold. Indeed, there are pathological conditions, such as malignant hyperthermia, in which control is lost and both pathways proceed rapidly with the concomitant generation of heat by the rapid, uncontrolled hydrolysis of ATP.

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Q.1- Justify the statement -”Gluconeogenesis is not a reversal of glycolysis”.

Or

Give a brief account of the thermodynamic barriers of gluconeogenesis.

Answer- Gluconeogenesis is the process of converting noncarbohydrate precursors to glucose or glycogen. The major substrates are the glucogenic amino acids, lactate, glycerol, and propionate. These noncarbohydrate precursors of glucose are first converted into pyruvate or enter the pathway at later intermediates such as oxaloacetate and Dihydroxyacetone phosphate. Liver and kidney are the major gluconeogenic tissues.

Gluconeogenesis meets the needs of the body for glucose when sufficient carbohydrate is not available from the diet or glycogen reserves. A supply of glucose is necessary especially for the nervous system and erythrocytes. Failure of gluconeogenesis is usually fatal.

Reactions of Gluconeogenesis

Thermodynamic barriers

In glycolysis, glucose is converted into pyruvate; in gluconeogenesis, pyruvate is converted into glucose. However, gluconeogenesis is not a reversal of glycolysis. Three nonequilibrium reactions in glycolysis catalyzed by hexokinase, phosphofructokinase and pyruvate kinase are considered thermodynamic barriers which prevent simple reversal of glycolysis for glucose synthesis.

Several reactions must differ because the equilibrium of glycolysis lies far on the side of pyruvate formation. The actual ∆G for the formation of pyruvate from glucose is about -20 kcal mol-1 (-84 kJ mol-1) under typical cellular conditions. Most of the decrease in free energy in glycolysis takes place in the three essentially irreversible steps catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase.

 

 

 

 

 

 

 

 

 

In gluconeogenesis, the following new steps bypass these virtually irreversible reactions of glycolysis:

1. First bypass (Formation of Phosphoenolpyruvate from pyruvate)

Reversal of the reaction catalyzed by pyruvate kinase in glycolysis involves two endothermic reactions. Phosphoenolpyruvate is formed from pyruvate by way of oxaloacetate through the action of pyruvate carboxylase and phosphoenolpyruvate carboxykinase.

Mitochondrial pyruvate carboxylase catalyzes the carboxylation of pyruvate to oxaloacetate, an ATP-requiring reaction in which the vitamin biotin is the coenzyme. Biotin binds CO2 from bicarbonate as carboxybiotin prior to the addition of the CO2 to pyruvate.

 

 

 

Pyruvate carboxylase is a mitochondrial enzyme, whereas the other enzymes of gluconeogenesis are cytoplasmic.

Oxaloacetate, the product of the pyruvate carboxylase reaction, is reduced to malate inside the mitochondrion for transport to the cytosol. The reduction is accomplished by an NADH-linked malate dehydrogenase. When malate has been transported across the mitochondrial membrane, it is reoxidized to oxaloacetate by an NAD+-linked malate dehydrogenase in the cytosol.

 

 

 

 

 

 

 

 

 

Figure-1- showing the transportation of oxaloacetate outside the mitochondrion in the form of Malate

A second enzyme, phosphoenolpyruvate carboxykinase, catalyzes the decarboxylation and phosphorylation of oxaloacetate to phosphoenolpyruvate using GTP as the phosphate donor. In liver and kidney, the reaction of succinate thiokinase in the citric acid cycle produces GTP (rather than ATP as in other tissues), and this GTP is used for the reaction of phosphoenolpyruvate carboxykinase, thus providing a link between citric acid cycle activity and gluconeogenesis, to prevent excessive removal of oxaloacetate for gluconeogenesis, which would impair citric acid cycle activity.

 

 

 

 

2. Second bypass (Formation of Fructose 6-phosphate is formed from fructose 1,6-bisphosphate)

On formation, phosphoenolpyruvate is metabolized by the enzymes of glycolysis but in the reverse direction. These reactions are near equilibrium under intracellular conditions; so, when conditions favor gluconeogenesis, the reverse reactions will take place until the next irreversible step is reached. This step is the hydrolysis of fructose 1,6- bisphosphate to fructose 6-phosphate and Pi.

 

 

 

 

Fructose 1,6-bisphosphatase catalyzes this exergonic hydrolysis.

Its presence determines whether a tissue is capable of synthesizing glucose (or glycogen) not only from pyruvate, but also from triose phosphates. It is present in liver, kidney, and skeletal muscle, but is probably absent from heart and smooth muscle. Like its glycolytic counterpart, it is an allosteric enzyme that participates in the regulation of gluconeogenesis.

3. Third bypass (Formation of Glucose by hydrolysis of glucose 6-phosphate)

The fructose 6-phosphate generated by fructose 1,6-bisphosphatase is readily converted into glucose 6-phosphate. In most tissues, gluconeogenesis ends here. Free glucose is not generated; rather, the glucose 6-phosphate is processed in some other fashion, notably to form glycogen. One advantage to ending gluconeogenesis at glucose 6-phosphate is that, unlike free glucose, the molecule cannot diffuse out of the cell. To keep glucose inside the cell, the generation of free glucose is controlled in two ways. First, the enzyme responsible for the conversion of glucose 6-phosphate into glucose, glucose 6-phosphatase, is regulated.

 

 

 

Second, the enzyme is present only in tissues whose metabolic duty is to maintain blood-glucose homeostasis- tissues that release glucose into the blood. These tissues are the liver and to a lesser extent the kidney the enzyme is absent in muscle and adipose tissue, which therefore, cannot export glucose into the bloodstream.

 

 

 

 

 

 

 

 

 

 

Figure-2- The glucose 6-phosphatase enzyme is stabilized by the Ca2+-binding protein SP .After the cleavage, a set of transporters, T2 and T3, return the products orthophosphate and glucose back into the cytosol.

This final step in the generation of glucose does not take place in the cytosol. Rather, glucose 6-phosphate is transported into the lumen of the endoplasmic reticulum, where it is hydrolyzed to glucose by glucose 6-phosphatase, which is bound to the membrane .An associated Ca2+-binding stabilizing protein is essential for phosphatase activity. Glucose and Pi are then shuttled back to the cytosol by a pair of transporters. The glucose transporter in the endoplasmic reticulum membrane is like those found in the plasma membrane. It is striking that five proteins are needed to transform cytosolic glucose 6-phosphate into glucose.

In the kidney, muscle and especially the liver, G6P be shunted toward glycogen if blood glucose levels are adequate. The reactions necessary for glycogen synthesis are an alternate bypass series of reactions.

The G6P produced from gluconeogenesis can be converted to glucose-1-phosphate (G1P) by phosphoglucose mutase (PGM). G1P is then converted to UDP-glucose (the substrate for glycogen synthase) by UDP-glucose pyro phosphorylase, a reaction requiring hydrolysis of UTP.

 

 

 

 

                               

 

 

 

 

 

 

 

 

 

 

 

Figure-3- showing reactions of gluconeogenesis

Energetics of gluconeogenesis

 The overall reaction of gluconeogenesis is-

 

 

 

The overall reaction of glycolysis is-

 

 

 

 

Six nucleotide triphosphate molecules are hydrolyzed to synthesize glucose from pyruvate in gluconeogenesis, whereas only two molecules of ATP are generated in glycolysis in the conversion of glucose into pyruvate.  Thus it is not a simple reversal of glycolysis but it is energetically an expensive affair.

The four additional high phosphoryl-transfer potential molecules are needed to turn an energetically unfavorable process (the reversal of glycolysis, D G°´ = + 20 kcal mol-1 [+84 kJ mol-1]) into a favorable one (gluconeogenesis, D G°´ = -9 kcal mol-1 [-38 kJ mol-1]). This is a clear example of the coupling of reactions: ATP hydrolysis is used to power an energetically unfavorable reaction.

 Q.2- How do the substrates of gluconeogenesis gain entry in to the main pathway?

Explain giving reactions involving each substrate.

Answer- The major substrates are the glucogenic amino acids, lactate, glycerol, and propionate.

A) Glucogenic amino acids- Amino acids are derived from the dietary proteins, tissue proteins or from the breakdown of skeletal muscle proteins during starvation. After transamination or deamination, glucogenic amino acids yield either pyruvate or intermediates of the citric acid cycle. Amino acids that are degraded to acetyl CoA or Acetoacetyl CoA are termed ketogenic amino acids because they can give rise to ketone bodies or fatty acids. Amino acids that are degraded to pyruvate, α-ketoglutarate, succinyl CoA, fumarate, or oxaloacetate are termed glucogenic amino acids. The net synthesis of glucose from these amino acids is feasible because these citric acid cycle intermediates and pyruvate can be converted into phosphoenolpyruvate. The entry point of these glucogenic amino acids in to the pathway of gluconeogenesis is as follows-

1) Pyruvate is the point of entry for alanine, serine, cysteine, glycine, Threonine, and tryptophan (Figure-4). The transamination of alanine directly yields pyruvate.

 

 

2) Oxalo acetate- Aspartate and asparagine are converted into oxaloacetate, a citric acid cycle intermediate. Aspartate, a four-carbon amino acid, is directly transaminated to oxaloacetate.

 

 

3) α-Ketoglutarate is the point of entry of several five-carbon amino acids that are first converted into glutamate.(Figure)

4) Succinyl CoA is a point of entry for some of the carbon atoms of methionine, isoleucine, and valine. Propionyl CoA and then Methylmalonyl CoA are intermediates in the breakdown of these three nonpolar amino acids.

5) Fumarate is the point of entry for Aspartate, Phenyl alanine and Tyrosine.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure- 4- Amino acids forming  Acetyl co A or Acetoacetyl co A are not considered glucogenic, they are called ketogenic amino acids since acetyl co A is a precursor for ketone bodies. All other amino acids which form pyruvate or intermediates of TCA cycle are considered glucogenic.

 B) Lactate- Lactate is formed by active skeletal muscle when the rate of glycolysis exceeds the rate of oxidative metabolism. Lactate is readily converted into pyruvate by the action of lactate dehydrogenase.

 

 

 

 

 

 

 

Lactate is a major source of carbon atoms for glucose synthesis by gluconeogenesis. During anaerobic glycolysis in skeletal muscle, pyruvate is reduced to lactate by lactate dehydrogenase (LDH). This reaction serves two critical functions during anaerobic glycolysis. First, in the direction of lactate formation the LDH reaction requires NADH and yields NAD+ which is then available for use by the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis. These two reactions are, therefore, intimately coupled during anaerobic glycolysis. Secondly, the lactate produced by the LDH reaction is released to the blood stream and transported to the liver where it is converted to glucose. The glucose is then returned to the blood for use by muscle as an energy source and to replenish glycogen stores. This cycle is termed the Cori cycle.(See details)

C) Propionate- Propionate is a major precursor of glucose in ruminants; it enters gluconeogenesis via the citric acid cycle. After esterificaton with CoA, Propionyl-CoA is carboxylated to D-Methylmalonyl-CoA, catalyzed by Propionyl-CoA carboxylase, a biotin-dependent enzyme (Figure-5). Methylmalonyl-CoA racemase catalyzes the conversion of D-Methylmalonyl-CoA to L-Methylmalonyl-CoA, which then undergoes isomerization to succinyl-CoA catalyzed by Methylmalonyl-CoA mutase. In non-ruminants, including humans, propionate arises from the Beta -oxidation of odd-chain fatty acids that occur in ruminant lipids, as well as the oxidation of isoleucine and the side-chain of cholesterol, and is a (relatively minor) substrate for gluconeogenesis. Methylmalonyl CoA Isomerase/ mutase is a vitamin B12 dependent enzyme, and in deficiency methylmalonic acid is excreted in the urine (methylmalonic aciduria). (Figure-5)

 

 

 

 

 

 

 

 

 

 

 

 

Figure-5- showing the fate of Propionyl co A

D) Glycerol-The hydrolysis of triacylglycerols in fat cells yields glycerol and fatty acids. Glycerol may enter either the gluconeogenic or the glycolytic pathway at Dihydroxyacetone phosphate; however, the carbons of the fatty acids cannot be utilized for net synthesis of glucose. In the fasting state glycerol released from lipolysis of adipose tissue triacylglycerol is used solely as a substrate for gluconeogenesis in the liver and kidneys. This requires phosphorylation to glycerol-3-phosphate by glycerol kinase and dehydrogenation to Dihydroxyacetone phosphate (DHAP) by glyceraldehyde-3-phosphate dehydrogenase (G3PDH). The G3PDH reaction is the same as that used in the transport of cytosolic reducing equivalents into the mitochondrion for use in oxidative phosphorylation. This transport pathway is called the glycerol-phosphate shuttle.

 

 

 

 

 

 

 

Figure-6- showing the conversion of glycerol to dihydroxy acetone phosphate

Glycerol kinase is absent in adipose tissue, so glycerol released by hydrolysis of triglycerides  can not be utilized for re esterificaton, it is a waste product, It is carried through circulation to the liver and is used for gluconeogenesis or glycolysis as the need may be. In fact adipocytes require a basal level of glycolysis in order to provide them with DHAP as an intermediate in the synthesis of triacylglycerols.

Q.3-. “It is incorrect to say that fats can not be converted to glucose”, Comment on the statement.

Answer- It is a justified statement.

Triglycerides (fats) on hydrolysis yield fatty acids and glycerol.Even chain fatty acids are not the glucogenic precursors, Oxidation of these fatty acids yields enormous amounts of energy on a molar basis, however, the carbons of the fatty acids cannot be utilized for net synthesis of glucose. The two carbon unit of acetyl-CoA derived from β-oxidation of fatty acids can be incorporated into the TCA cycle, however, during the TCA cycle two carbons are lost as CO2. Moreover the formation of acetyl CoA from pyruvate is an irreversible step, thus acetyl CoA can not be converted back into glucose. The oxidative decarboxylation of pyruvate to acetyl CoA commits the carbon atoms of glucose to two principal fates: oxidation to CO2 by the citric acid cycle, with the concomitant generation of energy, or incorporation into lipid (Figure-7),  Thus, explaining why even chain fatty acids do not undergo net conversion to carbohydrate.

Odd chain fatty acids on oxidation produce Propionyl co A which is a substrate for gluconeogenesis through formation of succinyl co A.

Glycerol component of fats can also be utilized for the formation of glucose through formation of dihydroxy acetone phosphate. Hence therefore except for even chain fatty acids, the other fat components are glucogenic, so the above given statement that “It is incorrect to say that fats can not be converted to glucose”, is a justified statement.

 

 

 

 

 

 

 

 

 

 

Figure-7- showing the irreversible reaction of Pyruvate to Acetyl co A

Q.4- What is Cori cycle or Lactic acid cycle? How does it differ from Glucose Alanine cycle? Explain how muscle may be used to supply energy in a wasting condition like anorexia nervosa.

Answer- Lactate produced by active skeletal muscle and erythrocytes is a source of energy for other organs. Erythrocytes lack mitochondria and can never oxidize glucose completely. In contracting skeletal muscle during vigorous exercise, the rate at which glycolysis produces pyruvate exceeds the rate at which the citric acid cycle oxidizes it. Under these conditions, moreover, the rate of formation of NADH by glycolysis is greater than the rate of its oxidation by aerobic metabolism.

Continued glycolysis depends on the availability of NAD+ for the oxidation of glyceraldehyde 3-phosphate. The accumulation of both NADH and pyruvate is reversed by lactate dehydrogenase, which oxidizes NADH to NAD+ as it reduces pyruvate to lactate. However, lactate is a dead-end in metabolism. It must be converted back into pyruvate before it can be metabolized. The only purpose of the reduction of pyruvate to lactate is to regenerate NAD+ so that glycolysis can proceed in active skeletal muscle and erythrocytes. The formation of lactate buys time and shiftspart of the metabolic burden from muscle to other organs.

 

 

 

 

 

 

 

 

Figure-8- showing Cori cycle

The plasma membrane of most cells contains carriers that render them highly permeable to lactate and pyruvate. Both substances diffuse out of active skeletal muscle into the blood and are carried to the liver. Much more lactate than pyruvate is transported out because the high NADH/NAD+ ratio in contracting skeletal muscle favors the conversion of pyruvate into lactate. The lactate that enters the liver is oxidized to pyruvate, a reaction favored by the low NADH/NAD+ ratio in the cytosol of liver cells. Pyruvate in the liver is converted into glucose by the gluconeogenic pathway. Glucose then enters the blood and is taken up by skeletal muscle. Thus, the liver furnishes glucose to contracting skeletal muscle, which derives ATP from the glycolytic conversion of glucose into lactate. Contracting skeletal muscle supplies lactate to the liver, which uses it to synthesize glucose. These reactions constitute the Cori cycle (Figure-7).

Glucose Alanine cycle

The glucose-alanine cycle is used primarily as a mechanism for skeletal muscle to eliminate nitrogen while replenishing its energy supply. Glucose oxidation produces pyruvate which can undergo transamination to alanine. This reaction is catalyzed by alanine transaminase, ALT (ALT used to be referred to a serum glutamate-pyruvate transaminase, SGPT). Additionally, during periods of fasting, skeletal muscle protein is degraded for the energy value of the amino acid carbons and alanine is a major amino acid in protein. The alanine then enters the blood stream and is transported to the liver. Within the liver alanine is converted back to pyruvate which is then a source of carbon atoms for gluconeogenesis. The newly formed glucose can then enter the blood for delivery back to the muscle. The amino group transported from the muscle to the liver in the form of alanine is converted to urea in the urea cycle and excreted.(Figure-9)

Role of Muscle in wasting conditions, starvation or in Anorexia nervosa

Plasma glucose concentrations are normally maintained within a relatively narrow range, roughly 70–110 mg/dL (3.9–6.1 mmol/L) in the fasting state with transient higher excursions after a meal, despite wide variations in exogenous glucose delivery from meals and in endogenous glucose utilization by, for example, exercising muscle. Between meals and during fasting, plasma glucose levels are maintained by endogenous glucose production, hepatic glycogenolysis, and hepatic (and renal) gluconeogenesis. Although hepatic glycogen stores are usually sufficient to maintain plasma glucose levels for approximately 8 h, this time period can be shorter if glucose demand is increased by exercise or if glycogen stores are depleted by illness or starvation.

Gluconeogenesis requires a coordinated supply of precursors from muscle and adipose tissue to the liver (and kidneys). Muscle provides lactate, pyruvate, Alanine, glutamine, and other amino acids. Triglycerides in adipose tissue are broken down into fatty acids and glycerol, which is a gluconeogenic precursor. Fatty acids provide an alternative oxidative fuel to tissues other than the brain (which requires glucose).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure- 9-showing glucose- alanine cycle

This glucose-alanine cycle thus provides an indirect way of utilizing muscle glycogen to maintain blood glucose in the fasting state. The ATP required for the hepatic synthesis of glucose from pyruvate is derived from the oxidation of fatty acids. Thus by interplay of glycolysis and Gluconeogenesis, the energy requirements of different cell types are fulfilled.

 

 

 

 

 

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Q.1-What is TCA cycle? Describe the steps and explain why can’t citric acid cycle operate in the absence of oxygen?

Answer-The citric acid cycle is the central metabolic hub of the cell. It is the final common pathway for the oxidation of fuel molecule such as amino acids, fatty acids, and carbohydrates.It is the gateway to the aerobic metabolism of any molecule that can be transformed into an acetyl group or dicarboxylic acid.

The cycle is also an important source of precursors, not only for the storage forms of fuels, but also for the building blocks of many other molecules such as amino acids, nucleotide bases, cholesterol, and porphyrin (the organic component of heme).

In eukaryotes, the reactions of the citric acid cycle take place inside mitochondria, in contrast with those of glycolysis, which take place in the cytosol.

An Overview of the Citric Acid Cycle

The citric acid cycle (Krebs cycle, tricarboxylic acid cycle) includes a series of oxidation-reduction reactions in mitochondria that result in the oxidation of an acetyl group to two molecules of carbon dioxide and reduce the coenzymes that are reoxidized through the electron transport chain, linked to the formation of ATP.

A four- carbon compound (oxaloacetate) condenses with a two-carbon acetyl unit to yield a six-carbon tricarboxylic acid (citrate). An isomer of citrate is then oxidatively decarboxylated. The resulting five-carbon compound (α-ketoglutarate) also is oxidatively decarboxylated to yield a four carbon compound (succinate).(Figure-1)

                                 

  

 

 

 

 

 

 

 

 

Figure-1- showing overview of TCA cycle

Oxaloacetate is then regenerated from succinate. Two carbon atoms enter the cycle as an acetyl unit and two carbon atoms leave the cycle in the form of two molecules of carbon dioxide. Three hydride ions (hence, six electrons) are transferred to three molecules of nicotinamide adenine dinucleotide (NAD+), whereas one pair of hydrogen atoms (hence, two electrons) are transferred to one molecule of flavin adenine dinucleotide (FAD) (Figure-2).

The function of the citric acid cycle is the harvesting of high-energy electrons from carbon fuels.

The citric acid cycle itself neither generates a large amount of ATP nor includes oxygen as a reactant. Instead, the citric acid cycle removes electrons from acetyl CoA and uses these electrons to form NADH and FADH2 (Figure-2). In oxidative phosphorylation, electrons released in the reoxidation of NADH and FADH2 flow through a series of membrane proteins (referred to as the electron-transport chain) to generate a proton gradient across the membrane.

These protons then flow through ATP synthase to generate ATP from ADP and inorganic phosphate.

 

 

 

 

 

 

 

 

 

 

 

 

Figure- 2-showing function of the citric acid cycle in transforming fuel molecules into ATP. fuel molecules are carbon compounds that are capable of being oxidized (of losing electrons)

Requirement of oxygen- Oxygen is required for the citric acid cycle indirectly inasmuch as it is the electron acceptor at the end of the electron-transport chain, necessary to regenerate NAD+ and FAD. The citric acid cycle, in conjunction with oxidative phosphorylation, provides the vast majority of energy used by aerobic cells in human beings, greater than 95%.

The four-carbon molecule, oxaloacetate that initiates the first step in the citric acid cycle is regenerated at the end of one passage through the cycle. The oxaloacetate acts catalytically: it participates in the oxidation of the acetyl group but is itself regenerated. Thus, one molecule of oxaloacetate is capable of participating in the oxidation of many acetyl molecules.

Reactions of the Citric Acid Cycle

The enzymes of the citric acid cycle are located in the mitochondrial matrix, either free or attached to the inner mitochondrial membrane and the crista membrane, where the enzymes of the respiratory chain are also found.

1) Formation of Citrate- The citric acid cycle begins with the condensation of a four-carbon unit, oxaloacetate, and a two-carbon unit, the acetyl group of acetyl CoA. Oxaloacetate reacts with acetyl CoA and H2O to yield citrate and CoA. This reaction, which is an aldol condensation followed by a hydrolysis, is catalyzed by citrate synthase. Oxaloacetate first condenses with acetyl CoA to form citryl CoA, which is then hydrolyzed to citrate and CoA. The hydrolysis of citryl CoA, a high-energy thioester intermediate, drives the overall reaction far in the direction of the synthesis of citrate. In essence, the hydrolysis of the thioester powers the synthesis of a new molecule from two precursors.

 

 

 

 

 

 

 

 

Figure-3- showing the formation of citrate, citrate synthase catalyzes this reaction

 2) Formation of Isocitrate- The tertiary hydroxyl group is not properly located in the citrate molecule for the oxidative decarboxylation that follows. Thus, citrate is isomerized into isocitrate to enable the six-carbon unit to undergo oxidative decarboxylation. The isomerization of citrate is accomplished by a dehydration step followed by a hydration step. The result is an interchange of a hydrogen atom and a hydroxyl group. The enzyme catalyzing both steps is called Aconitase because cis-aconitate is an intermediate.

 

 

 

 

 

 

 

 

 

Figure-4- showing formation of isocitrate

 

Aconitase is an iron-sulfur protein, or nonheme iron protein. It contains four iron atoms that are not incorporated as part of a heme group. The four iron atoms are complexed to four inorganic sulfides and three cysteine sulfur atoms, leaving one iron atom available to bind citrate and then isocitrate through their carboxylate and hydroxyl groups. This iron center, in conjunction with other groups on the enzyme, facilitates the dehydration and rehydration reactions.

The poison Fluoroacetate is toxic, because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits Aconitase, causing citrate to accumulate.

 3) Formation of α- Keto Glutarate

Isocitrate undergoes dehydrogenation catalyzed by isocitrate dehydrogenase to form, initially,Oxalo succinate, which remains enzyme-bound and undergoes decarboxylation to α -ketoglutarate. The decarboxylation requires Mg++ or Mn++ ions. There are three isoenzymes of isocitrate dehydrogenase. One, which uses NAD+, is found only in mitochondria. The other two use NADP+ and are found in mitochondria and the cytosol. Respiratory chain-linked oxidation of isocitrate proceeds almost completely through the NAD+-dependent enzyme.

 

 

 

 

 

 

 

 

Figure-5- showing the formation of α- Keto Glutarate from Isocitrate

 4) Formation of Succinyl Co A

The conversion of isocitrate into α-ketoglutarate is followed by a second oxidative decarboxylation reaction, the formation of succinyl CoA from α–ketoglutarate.

 

 

 

 

 

 

 

 

Figure-6- showing the formation of Succinyl co A

 

α-Ketoglutarate undergoes oxidative decarboxylation in a reaction catalyzed by a multi-enzyme complex similar to that involved in the oxidative decarboxylation of pyruvate. The α--ketoglutarate dehydrogenase complex requires the same cofactors as the pyruvate dehydrogenase complex—thiamin diphosphate, lipoate, NAD+, FAD, and CoA—and results in the formation of succinyl-CoA. The equilibrium of this reaction is so much in favor of succinyl-CoA formation that it must be considered to be physiologically unidirectional. As in the case of pyruvate oxidation, arsenite inhibits the reaction, causing the substrate, α -ketoglutarate, to accumulate.

 5) Formation of Succinate- Succinyl CoA is an energy-rich thioester compound. The ∆for the hydrolysis of succinyl CoA is about -8 kcal mol-1 (-33.5 kJ mol-1), which is comparable to that of ATP (-7.3 kcal mol-1, or -30.5 kJ mol-1). The cleavage of the thioester bond of succinyl CoA is coupled to the phosphorylation of a purine nucleoside diphosphate, usually GDP. This reaction is catalyzed by succinyl CoA synthetase (succinate thiokinase).

 

 

 

 

 

 

 

 

Figure-7- showing the formation of succinate

This is the only example in the citric acid cycle of substrate level phosphorylation. Tissues in which gluconeogenesis occurs (the liver and kidney) contain two isoenzymes of succinate thiokinase, one specific for GDP and the other for ADP. The GTP formed is used for the decarboxylation of oxaloacetate to phosphoenolpyruvate in gluconeogenesis, and provides a regulatory link between citric acid cycle activity and the withdrawal of oxaloacetate for gluconeogenesis. Nongluconeogenic tissues have only the isoenzyme that uses ADP.

6) Regeneration of Oxaloacetate

The onward metabolism of succinate, leading to the regeneration of oxaloacetate, is the same sequence of chemical reactions as occurs in the β-oxidation of fatty acids: dehydrogenation to form a carbon-carbon double bond, addition of water to form a hydroxyl group, and a further dehydrogenation to yield the oxo-group of oxaloacetate.

The first dehydrogenation reaction, forming fumarate, is catalyzed by succinate dehydrogenase, which is bound to the inner surface of the inner mitochondrial membrane. The enzyme contains FAD and iron-sulfur (Fe:S) protein, and directly reduces ubiquinone in the electron transport chain.

 

 

 

FAD is the hydrogen acceptor in this reaction because the free-energy change is insufficient to reduce NAD+.

Fumarase (fumarate hydratase) catalyzes the addition of water across the double bond of fumarate, yielding malate.

 

 

 

 

 

 

 

 

 

 

Figure-8- showing the formation of Malate from Fumarate

Malate is converted to oxaloacetate by malate dehydrogenase, a reaction requiring NAD+.

 

 

 

Although the equilibrium of this reaction strongly favors malate, the net flux is to oxaloacetate because of the continual removal of oxaloacetate (to form citrate, as a substrate for gluconeogenesis, or to undergo transamination to aspartate) and also the continual reoxidation of NADH.

 

 

 

 

 

 

 

 

Figure-9- showing the regeneration of Oxaloacetate from Succinate

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure- 10-showing the reactions of TCA cycle, The cycle starts with the condensation of Acetyl co A with oxaloacetate, which is regenerated at the end of the cycle. Thus oxaloacetate acts as a catalyst for the cycle.

 

 Energy yield per Acetyl co A per turn of cycle

The net reaction of the citric acid cycle is-

 

 

 

 

As a result of oxidations catalyzed by the dehydrogenases of the citric acid cycle, three molecules of NADH and one of FADH2 are produced for each molecule of acetyl-CoA catabolized in one turn of the cycle. These reducing equivalents are transferred to the respiratory chain, where reoxidation of each NADH results in formation of 3, and 2 ATP of FADH2. Consequently, 11 high-transfer-potential phosphoryl groups are generated when the electron-transport chain oxidizes 3 molecules of NADH and 1 molecule of FADH2,  In addition, 1 ATP (or GTP) is formed by substrate-level phosphorylation catalyzed by succinate thiokinase.

Thus, 1 acetate unit generates approximately 12 molecules of ATP. In dramatic contrast, only 2 molecules of ATP are generated per molecule of glucose (which generates 2 molecules of acetyl CoA) by anaerobic glycolysis.

Molecular oxygen does not participate directly in the citric acid cycle. However, the cycle operates only under aerobic conditions because NAD+ and FAD can be regenerated in the mitochondrion only by the transfer of electrons to molecular oxygen. Glycolysis has both an aerobic and an anaerobic mode, whereas the citric acid cycle is strictly aerobic. Glycolysis can proceed under anaerobic conditions because NAD+ is regenerated in the conversion of pyruvate into lactate.

Q.2- What is the total energy yield when glucose is completely oxidized to CO2 and water?

 Answer-

Oxidation of Glucose yields up to 38 Mol of ATP under aerobic conditions, but only 2 Mol when O2 is absent

When 1 mol of glucose is combusted in a calorimeter to CO2 and water, approximately 2870 kJ are liberated as heat. When oxidation occurs in the tissues, approximately 38 mol of ATP are generated per molecule of glucose oxidized to CO2 and water. In vivo, ∆G for the ATP synthase reaction has been calculated as approximately 51.6 kJ. It follows that the total energy captured in ATP per mole of glucose oxidized is 1761 kJ, or approximately 68% of the energy of combustion. Most of the ATP is formed by oxidative phosphorylation resulting from the reoxidation of reduced coenzymes by the respiratory chain. The remainder is formed by substrate level phosphorylation.

ATP Formation in the Catabolism of Glucose

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

This assumes that NADH formed in glycolysis is transported into mitochondria by the malate shuttle .If the Glycerophosphate shuttle is used, then only 2 ATP will be formed per mol of NADH.

There is a considerable advantage in using glycogen rather than glucose for anaerobic glycolysis in muscle, since the product of glycogen phosphorylase is glucose 1-phosphate, which is interconvertible with glucose 6-phosphate. This saves the ATP that would otherwise be used by hexokinase, increasing the net yield of ATP from 2 to 3 per glucose.

 

Q.3- Discuss the regulation of TCA cycle.

Or

 What is respiratory control of TCA cycle?

Answer- Regulation of the TCA cycle like that of glycolysis occurs at both the level of entry of substrates into the cycle as well as at the key reactions of the cycle. Fuel enters the TCA cycle primarily as acetyl-CoA. The generation of acetyl-CoA from carbohydrates is, therefore, a major control point of the cycle. This is the reaction catalyzed by the PDH complex.

1) Regulation of PDH Complex- PDH complex is inhibited by acetyl-CoA and NADH and activated by non-acetylated CoA (CoASH) and NAD+. The pyruvate dehydrogenase activities of the PDH complex are regulated by their state of phosphorylation. This modification is carried out by a specific kinase (PDH kinase) and the phosphates are removed by a specific phosphatase (PDH phosphatase). The phosphorylation of PDH inhibits its activity and, therefore, leads to decreased oxidation of pyruvate. PDH kinase is activated by NADH and acetyl-CoA and inhibited by pyruvate, ADP, CoASH, Ca2+ and Mg2+. The PDH phosphatase, in contrast, is activated by Mg2+ and Ca2+.In a tissue such as brain, which is largely dependent on carbohydrate to supply acetyl-CoA, control of the citric acid cycle may occur at pyruvate dehydrogenase.

2)  Regulation of TCA cycle enzymes-In addition, individual enzymes of the cycle are regulated. The most likely sites for regulations are the nonequilibrium reactions catalyzed citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase. The dehydrogenases are activated by Ca2+, which increases in concentration during muscular contraction and secretion, when there is increased energy demand.

a) Citrate synthase-  There is allosteric inhibition of citrate synthase by ATP and long-chain fatty acyl-CoA.

b) Isocitrate dehydrogenase-  is allosterically stimulated by ADP, which enhances the enzyme’s affinity for substrates. In contrast, NADH inhibits iso-citrate dehydrogenase by directly displacing NAD+. ATP, too, is inhibitory.

c) α-ketoglutarate dehydrogenase -α- Ketoglutarate dehydrogenase is inhibited by succinyl CoA and NADH, the products of the reaction that it catalyzes. In addition, α-ketoglutarate dehydrogenase is inhibited by a high energy charge. Thus, the rate of the cycle is reduced whenthe cell has a high level of ATP.

d) Succinate dehydrogenase is inhibited by oxaloacetate, and the availability of oxaloacetate, as controlled by malate dehydrogenase, depends on the [NADH]/[NAD+] ratio.

Since three reactions of the TCA cycle as well as PDH utilize NAD+ as co-factor it is not difficult to understand why the cellular ratio of NAD+/NADH has a major impact on the flux of carbon through the TCA cycle. Thus, activity of TCA cycle is immediately dependent on the supply of NAD+, which in turn, because of the tight coupling between oxidation and phosphorylation, is dependent on the availability of ADP and hence, ultimately on the rate of utilization of ATP in chemical and physical work. Thus, respiratory control via the respiratory chain and oxidative phosphorylation primarily regulates citric acid cycle activity.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-11- showing the regulation of TCA cycle. Excess of ATP depicts energy rich state of the cell, hence TCA cycle is inhibited while reverse occurs when the cell is in a low energy state with excess of ADP.

 

Q.4- What is the significance of TCA Cycle?

Answer- The citric acid cycle is not only a pathway for oxidation of two-carbon units, but is also a major pathway for interconversion of metabolites arising from transamination and deamination of amino acids, and providing the substrates for amino acid synthesis by transamination, as well as for gluconeogenesis and fatty acid synthesis. Because it functions in both oxidative and synthetic processes, it is amphibolic.

A) Catabolic role- The citric acid cycle is the final common pathway for the oxidation of carbohydrate, lipid, and protein because glucose, fatty acids, and most amino acids are metabolized to acetyl-CoA or intermediates of the cycle. The function of the citric acid cycle is the harvesting of high-energy electrons from carbon fuels.

1 acetate unit generates approximately 12 molecules of ATP per turn of the cycle.

 B) Anabolic role-As a major metabolic hub of the cell, the citric acid cycle also provides intermediates for biosynthesis of various compounds.

 i) Role in Gluconeogenesis- All the intermediates of the cycle are potentially glucogenic, since they can give rise to oxaloacetate, and hence net production of glucose (in the liver and kidney, the organs that carry out gluconeogenesis. The key enzyme that catalyzes net transfer out of the cycle into gluconeogenesis is phosphoenolpyruvate carboxykinase, which catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate, with GTP acting as the phosphate donor.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure- 12-showing the formation of Phosphoenolpyruvate from oxaloacetate, which can be subsequently used for the synthesis of glycine, Serine and Cysteine

Net transfer into the cycle occurs as a result of several reactions. Among the most important of such Anaplerotic reactions is the formation of oxaloacetate by the carboxylation of pyruvate, catalyzed by pyruvate carboxylase. This reaction is important in maintaining an adequate concentration of oxaloacetate for the condensation reaction with acetyl-CoA. If acetyl-CoA accumulates, it acts as both an allosteric activator of pyruvate carboxylase and an inhibitor of pyruvate dehydrogenase, thereby ensuring a supply of oxaloacetate.

Lactate, an important substrate for gluconeogenesis, enters the cycle via oxidation to pyruvate and then carboxylation to oxaloacetate.

Aminotransferase (transaminase) reactions form pyruvate from alanine, oxaloacetate from aspartate, and Alpha-ketoglutarate from glutamate. Other amino acids contribute to gluconeogenesis because their carbon skeletons give rise to citric acid cycle intermediates. Alanine, cysteine, glycine, hydroxyproline, serine, threonine, and tryptophan yield pyruvate; arginine, histidine, glutamine, and proline yield α-ketoglutarate; isoleucine, methionine, and valine yield succinyl-CoA; tyrosine and phenylalanine yield fumarate.

The conversion of propionate to succinyl-CoA via the Methylmalonyl-CoA pathway is also important for gluconeogenesis.

 ii) Role in synthesis of nonessential amino acids- Since the transamination reactions are reversible, the cycle also serves as a source of carbon skeletons for the synthesis of some amino acids like Alanine, aspartate, Asparagine Glutamate , glutamine etc. (Figure)

 


 
 

 

 

 

 

 

 

 

 

 

 

 

Figure-13- showing the formation of non essential amino acids from the TCA cycle intermediate. Glutamine  is subsequently utilized fro the synthesis of purine nucleotides

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure -14-showing the formation of non essential amino acids from the TCA cycle intermediate. Aspartic acid is subsequently utilized fro the synthesis of pyrimidine  nucleotides

 iii) Role in fatty acid synthesis- Acetyl-CoA, formed from pyruvate by the action of pyruvate dehydrogenase, is the major substrate for long-chain fatty acid synthesis . Pyruvate dehydrogenase is a mitochondrial enzyme, and fatty-acid synthesis is a cytosolic pathway; the mitochondrial membrane is impermeable to acetyl-CoA. Acetyl-CoA is made available in the cytosol from citrate synthesized in the mitochondrion, transported into the cytosol, and cleaved in a reaction catalyzed by ATP-citrate lyase.

 

 

 

 

 

 

 

 

 

 

 

 

Figure-15- showing transportation of citrate out of mitochondrion to provide Acetyl co A for fatty acid or cholesterol synthesis

Citrate is only available for transport out of the mitochondrion when Aconitase is saturated with its substrate, and citrate cannot be channeled directly from citrate synthase onto Aconitase. This ensures that citrate is used for fatty acid synthesis only when there is an adequate amount to ensure continued activity of the cycle. Acetyl co A can also be used for the synthesis of cholesterol, steroids etc.

iv)  Role in Haem synthesis- Succinyl co A condenses with amino acid Glycine to form Alpha amino beta keto adipic acid, which is the first step of haem biosynthesis.

v) Role in purine and pyrimidine synthesis- Glutamate and Aspartate derived from TCA cycle are utilized for the synthesis of purines and pyrimidines.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-16- showing the biosynthetic role of TCA cycle

  Anabolic Significance of individual intermediate

 1) Acetyl co A- It is a precursor for fatty acids, cholesterol, steroids, ketone bodies, acetyl choline and is also required for detoxification of xenobiotics.

2) Citrate- Citrate acts as transporter for export of Acetyl co A from mitochondria to cytoplasm for fatty acids and sterol synthesis.

3) Alpha ketoglutarate- forms a first link between TCA cycle and Nitrogen metabolism through formation of non essential amino acids , like Glutamate, Glutamine etc. Glutamine is required for the synthesis of purine and pyrimidines. Glutamate is  a precursor for GABA, which acts as a neurotransmitter.

4) Succinyl coA- is required for haem synthesis, utilization of ketone bodies, detoxification and itself acts as a neurotransmitter.

5) Fumarate- forms a link between TCA cycle and Urea cycle.

6) Oxaloacetate- acts as a substrate for glucose and  non essential amino acids. Aspartic acid produced from oxaloacetate is used for the synthesis of purines and pyrimidines.

Q.5.- Discuss the role played by vitamins in the operation of TCA cycle

 Answer- Vitamins play a key role in the working of TCA cycle. The following vitamins participate-

Five of the B vitamins are essential in the citric acid cycle and hence energy-yielding metabolism: (1) riboflavin, in the form of flavin adenine dinucleotide (FAD), a cofactor for succinate dehydrogenase; (2) niacin, in the form of nicotinamide adenine dinucleotide (NAD), the electron acceptor for isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase; (3) thiamin (vitamin B1), as thiamin diphosphate, the coenzyme for decarboxylation in the α -ketoglutarate dehydrogenase reaction; and (4) Pantothenic acid, as part of coenzyme A, the cofactor attached to “active” carboxylic acid residues such as acetyl-CoA and succinyl-CoA and (5) Biotin- in CO2 fixation reaction to compensate oxaloacetate concentration.

 Q6.- What are Anaplerotic reactions  ?

Or

How are citric acid cycle intermediates replenished if any are drawn off for biosyntheses?

Answer- Anaplerotic reactions (from the Greek Ana= ‘up’ and Plerotikos= ‘to fill’) are those that form intermediates of a metabolic pathway. Example of such can be the tricarboxylic acid (TCA) Cycle .In normal function of this cycle for respiration, concentrations of TCA intermediates remain constant; however, many biosynthetic reactions also use these molecules as a substrate. Anaplerosis is the act of replenishing TCA cycle intermediates that have been extracted for biosynthesis (in what are called cataplerotic reactions).

The TCA Cycle is a hub of metabolism, with central importance in both energy production and biosynthesis. Therefore, it is crucial for the cell to regulate concentrations of TCA Cycle metabolites in the mitochondria. Anaplerotic flux must balance cataplerotic flux in order to retain homeostasis of cellular metabolism

Reactions of Anaplerotic metabolism

There are 4 major reactions classed as Anaplerotic, yet the production of oxaloacetate from pyruvate has probably the most physiologic importance.

1) Formation of oxaloacetate from pyruvate- In case oxaloacetate is converted into amino acids for protein synthesis or used for gluneogenesis and, subsequently, the energy needs of the cell rise. The citric acid cycle will operate to a reduced extent unless new oxaloacetate is formed, because acetyl CoA cannot enter the cycle unless it condenses with oxaloacetate. Even though oxaloacetate is recycled, a minimal level must be maintained to allow the cycle to function.

How is oxaloacetate replenished? Mammals lack the enzymes for the net conversion of acetyl CoA into oxaloacetate or any other citric acid cycle intermediate. Rather, oxaloacetate is formed by –

a) The carboxylation of pyruvate, in a reaction catalyzed by the biotin-dependent enzyme pyruvate carboxylase.

This enzyme plays a crucial role in gluconeogenesis .It is active only in the presence of acetyl CoA, which signifies the need for more oxaloacetate. If the energy charge is high, oxaloacetate is converted into glucose.

If the energy charge is low, oxaloacetate replenishes the citric acid cycle.

b) Oxaloacetate can also be synthesized indirectly from Pyruvate through formation of Malate by Malic enzyme; Malate is subsequently converted to oxaloacetate by malate dehydrogenase enzyme.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-17- showing formation of oxaloacetate from pyruvate

 2)  Formation of oxaloacetate from Aspartate- Oxaloacetate can also be formed from Aspartate by transamination reaction.

 3)  Formation of Alpha ketoglutatarate-

Alpha ketoglutarate can be formed from Glutamate dehydrogenase or from transamination reactions.

 4) Formation of Succinyl co A – Succinyl co A can be produced from the oxidation of odd chain fatty acid and from the metabolism of methionine and isoleucine (through carboxylation of Propionyl co A to Methyl malonyl co A and then Succinyl co A)

 Since the citric acid cycle is a cycle, it can be replenished by the generation of any of the intermediates.

Q.7- Why is it said that fats burn in the flame of carbohydrates?

Answer- fats burn in the flame of carbohydrates means fats can only be oxidized in the presence of carbohydrates.

Acetyl co A represents Fat component, since the major source is fatty acid oxidation. Acetyl co A is completely oxidized in the TCA cycle in the presence of oxaloacetate. Pyruvate is mainly used up for Anaplerotic reactions to compensate for oxaloacetate concentration.  Thus without carbohydrates (Pyruvate), there would be no anaplerotic reactions to replenish the TCA-cycle components. With a diet of fats only, the acetyl CoA from fatty acid degradation would not get oxidized and build up due to non functioning of TCA cycle. Thus fats can burn only in the flame of carbohydrates.

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Q.1- Discuss the fate of pyruvate under different metabolic conditions.

Answer- The sequence of reactions from glucose to pyruvate is similar in most organisms and most types of cells. In contrast, the fate of pyruvate is variable. Three reactions of pyruvate are of prime importance: conversion into ethanol, lactic acid, or carbon dioxide

 1. Ethanol is formed from pyruvate in yeast and several other microorganisms. The first step is the decarboxylation of pyruvate. This reaction is catalyzed by pyruvate decarboxylase, which requires the coenzyme thiamine pyrophosphate.

This coenzyme, derived from the vitamin thiamine (B1), also participates in reactions catalyzed by other enzymes. The second step is the reduction of acetaldehyde to ethanol by NADH, in a reaction catalyzed by alcohol dehydrogenase.This process regenerates NAD+.

 

 

 

 

 

 

 

 

Figure-1- showing the conversion of pyruvate to Ethanol

The net result of this anaerobic process is:

 

 

 

 

The conversion of glucose into ethanol is an example of alcoholic fermentation. NADH generated by the oxidation of glyceraldehyde 3-phosphate is consumed in the reduction of acetaldehyde to ethanol. Thus, there is no net oxidation-reduction in the conversion of glucose into ethanol (Figure-1). The ethanol formed in alcoholic fermentation provides a key ingredient for brewing and wine making.

 2. Lactate is formed from pyruvate in a variety of microorganisms in a process called lactic acid fermentation. The reaction also takes place in the cells of higher organisms when the amount of oxygen is limiting, as in muscle during intense activity. The reduction of pyruvate by NADH to form lactate is catalyzed by lactate dehydrogenase.

 

 

 

 

 

 

Figure-2- showing the conversion of pyruvate to Lactate

 The overall reaction in the conversion of glucose into lactate is:

 

 

 

As in alcoholic fermentation, there is no net oxidation-reduction. The NADH formed in the oxidation of glyceraldehyde 3-phosphate is consumed in the reduction of pyruvate. The regeneration of NAD + in the reduction of pyruvate to lactateor ethanol sustains the continued operation of glycolysis under anaerobic conditions.

3. Acetyl co A – Only a fraction of the energy of glucose is released in its anaerobic conversion into ethanol or lactate. Much more energy can be extracted aerobically by means of the citric acid cycle and the electron-transport chain. The entry point to this oxidative pathway is acetyl coenzyme A (acetyl CoA), which is formed inside mitochondria by the oxidative decarboxylation of pyruvate.

 

 

 

 

The NAD+ required for this reaction and for the oxidation of glyceraldehyde 3-phosphate is regenerated when NADH ultimately transfers its electrons to O2 through the electron-transport chain in mitochondria. The reaction is catalyzed by a multi enzyme complex called Pyruvate dehydrogenase complex.

4. Oxaloacetate-  Pyruvate can be converted to oxaloacetate. Mitochondrial pyruvate carboxylase catalyzes the carboxylation of pyruvate to oxaloacetate, an ATP-requiring reaction in which the vitamin biotin is the coenzyme. Biotin binds CO2 from bicarbonate as carboxybiotin prior to the addition of the CO2 to pyruvate.

 

 

 

 

 

 

 

 

Figure –3- showing the conversion of pyruvate to oxaloacetate

 

The Oxaloacetate can be subsequently used for the synthesis of Aspartate, phosphoenol pyruvate or be utilized in the TCA cycle depending upon the need of the cell.

5. Alanine- Pyruvate can be transaminated to form Alanine as per need.

 

 

 

 

 

 

 

 

 

 

Figure-4- showing the conversion of Pyruvate to Alanine by transamination

This reaction is important for the catabolism and synthesis of non essential amino acids

6. Malate- Pyruvate can be directly converted to oxaloacetate or it is first carboxylated to malate and then decarboxylated to from oxaloacetate (Figure-5).  These two reactions are called CO2 filling up reactions or Anaplerotic reactions. They provide oxaloacetate when there is sudden influx of Acetyl co A in the TCA cycle.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-5- showing the formation of Oxalo acetate from Pyruvate.

 Thus Pyruvate can be metabolized through several pathways as per availability of O2 or requirement of the cell for a specific metabolite.

 Q.- 2-Give a brief account of Pyruvate dehydrogenase complex. How is this complex regulated? 

Answer- Under anaerobic conditions,  pyruvate is converted into lactic acid or ethanol, depending on the organism. Under aerobic conditions, the pyruvate is transported into mitochondria in exchange for OH- by the pyruvate carrier, an antiporter. In the mitochondrial matrix, pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex to form acetyl CoA.

 

 

 

This irreversible reaction is the link between glycolysis and the citric acid cycle. In the preparation of the glucose derivative pyruvate for the citric acid cycle, an oxidative decarboxylation takes place and high transfer- potential electrons in the form of NADH are captured.

Pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex is a large, highly integrated complex of three kinds of enzymes; Pyruvate dehydrogenase, dihydrolipoyl transacetylase and Dihydrolipoyl dehydrogenase. At least two additional enzymes regulate the activity of the complex and five coenzymes: thiamine pyrophosphate (TPP), lipoic acid, CoASH, FAD and NAD+ participate in the overall reaction. The pyruvate dehydrogenase complex consists of a number of polypeptide chains of each of the three component enzymes, and the intermediates do not dissociate, but remain bound to the enzymes.

Pyruvate dehydrogenase is a member of a family of homologous complexes that includes the citric acid cycle enzyme, alpha ketoglutarate dehydrogenase and for branched-chain  amino acids, alpha-ketoacid dehydrogenase,These complexes are large, with molecular masses ranging from 4 to 10 million daltons.

Reaction

The conversion of pyruvate into acetyl CoA consists of three steps: decarboxylation, oxidation, and transfer of the resultant acetyl group to CoA.

 

 

 

 

 

 

Figure-6 showing the processes involved in the conversion of pyruvate to Acetyl co A

These steps must be coupled to preserve the free energy derived from the decarboxylation step to drive the formation of NADH and acetyl CoA.

1)  Pyruvate is decarboxylated by the pyruvate dehydrogenase component of the enzyme complex to a hydroxyethyl derivative of the thiazole ring of enzyme-bound thiamin diphosphate,

2) Hydroxy ethyl TPP (Acyl TPP) reacts with oxidized lipoamide, the prosthetic group of dihydrolipoyl transacetylase, to form acetyl lipoamide.  In thiamin (vitamin B1 ) deficiency, glucose metabolism is impaired, and there is significant (and potentially life-threatening) lactic and pyruvic acidosis.

3)Acetyl lipoamide reacts with coenzyme A to form acetyl-CoA and reduced lipoamide.

4)The reaction is completed when the reduced lipoamide is reoxidized by a flavoprotein, dihydrolipoyl dehydrogenase, containing FAD.

5) Finally, the reduced flavoprotein is oxidized by NAD+, which in turn transfers reducing equivalents to the respiratory chain.

Each NADH yields 3 ATPS in the electron transport chain. Since from each Glucose two pyruvate molecules are produced, thus the net energy yield at this step is 6 ATPs.

(See figure-7)

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-7- Showing the reactions of pyruvate dehydrogenase comple,. During the oxidation of pyruvate to CO2 by pyruvate dehydrogenase the electrons flow from pyruvate to the lipoamide moiety of dihydrolipoyl transacetylase then to the FAD cofactor of dihydrolipoyl dehydrogenase and finally to reduction of NAD+ to NADH. The acetyl group is linked to coenzyme A (CoASH) in a high energy thioester bond. The acetyl-CoA then enters the TCA cycle for complete oxidation to CO2 and H2O.

Regulation of PDH complex

The reactions of the PDH complex serve to interconnect the metabolic pathways of glycolysis, gluconeogenesis and fatty acid synthesis to the TCA cycle. As a consequence, the activity of the PDH complex is highly regulated by a variety of allosteric effectors and by covalent modification.

Pyruvate dehydrogenase is inhibited by its products, acetyl-CoA and NADH.

It is also regulated by phosphorylation of three serine residues on the pyruvate dehydrogenase component of the multienzyme complex by a specific PDH kinase, resulting in decreased activity, and by dephosphorylation by a PDH phosphatase that causes an increase in activity

NADH and acetyl-CoA are powerful positive effectors on PDH kinase, the enzyme that inactivates PDH by converting it to the phosphorylated PDH-b form. The kinase is activated by increases in the [ATP]/[ADP], [acetyl-CoA]/[CoASH], and [NADH]/[NAD+] ratios. Since NADH and acetyl-CoA accumulate when the cell energy charge is high, it is not surprising that high ATP levels also up-regulate PDH kinase activity, reinforcing down-regulation of PDH activity in energy-rich cells. Note, however, that pyruvate is a potent negative effector on PDH kinase, with the result that when pyruvate levels rise, PDH-a will be favored even with high levels of NADH and acetyl-CoA.

Concentrations of pyruvate which maintain PDH in the active form (PDH-a) are sufficiently high so that, in energy-rich cells, the allosterically down-regulated, high Km form of PDH is nonetheless capable of converting pyruvate to acetyl-CoA. With large amounts of pyruvate in cells having high energy charge and high NADH, pyruvate carbon will be directed to the 2 main storage forms of carbon (glycogen via gluconeogenesis and fat production via fatty acid synthesis) where acetyl-CoA is the principal carbon donor.

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-8- showing the regulation of PDH complex

Although the regulation of PDH-b phosphatase is not well understood, it is likely to be regulated to maximize pyruvate oxidation under energy-poor conditions and to minimize PDH activity under energy-rich conditions. It is known that Mg2+ and Ca2+ activate the enzyme

Thus, pyruvate dehydrogenase, and therefore glycolysis, is inhibited both when there is adequate ATP (and reduced coenzymes for ATP formation) available, and also when fatty acids are being oxidized. In fasting, when free fatty acid concentrations increase, there is a decrease in the proportion of the enzyme in the active form, leading to a sparing of carbohydrate. In adipose tissue, glucose provides acetyl-CoA for lipogenesis, the enzyme is activated in response to insulin and in cardiac muscle PDH activity is increased by catecholamines.

 Q.3-What would be the consequences of Pyruvate dehydrogenase complex deficiency?

Answer- Pyruvate dehydrogenase complex deficiency (PDCD) is a rare disorder of carbohydrate metabolism caused by a deficiency of one or more enzymes in the pyruvate dehydrogenase complex. Dysfunctions in all 3 substrate-processing enzymes, regulatory proteins and thiamine dependence of the E1 alpha enzyme, have been described; however, dysfunction of the E1 alpha enzyme subunit is most common.

The age of onset and severity of disease depends on the activity level of the PDC enzymes. Individuals with PDCD beginning prenatally or in infancy usually die in early childhood. Those who develop PDCD later in childhood may have mental retardation and other neurological symptoms and usually survive into adulthood.

The following features are characteristic of this disease-

1) Energy Deficit-A deficiency in this enzymatic complex limits the production of citrate. Because citrate is the first substrate in the citric acid cycle, the cycle cannot proceed. Alternate metabolic pathways are stimulated in an attempt to produce acetyl-CoA; however, an energy deficit remains, especially in the CNS. The magnitude of the energy deficit depends on the residual activity of the enzyme.

2) Neurological deficit- Severe enzyme deficiencies may lead to congenital brain malformation because of a lack of energy during neural development. Underlying neuropathology is not usually observed in individuals whose onset of pyruvate dehydrogenase complex deficiency is in childhood.

The signs of poor neurological development or degenerative lesions are -Poor acquisition or loss of motor milestones, poor muscle tone, new onset seizures, and periods of incoordination (ie, ataxia) abnormal eye movements, poor response to visual stimuli, mental delay, psychomotor delays and growth retardation

High blood lactate and pyruvate levels with or without lactic acidemia suggest an inborn error of metabolism at the mitochondrial level.

Cofactor supplementation with thiamine, carnitine, and Lipoic acid is the standard of care. Ketogenic diets (with restricted carbohydrate intake) have been used to control lactic acidosis with minimal success.

Correction of acidosis does not reverse all the symptoms. CNS damage is common and limits recovery of normal function.

 

 

 

 

 

 

 

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Q.1- In mammals, glucose is the only fuel that the brain uses under non starvation conditions and the only fuel that red blood cells can use at all. There are many carbohydrates, Why is glucose instead of some other monosaccharides such a prominent fuel? 

Answer- First, glucose is one of the monosaccharides formed from formaldehyde under pre biotic conditions, so it may have been available as a fuel source for primitive biochemical systems.

Second, glucose has a low tendency, relative to other monosaccharides, to non enzymatically glycosylate proteins. In their open-chain (carbonyl) forms, monosaccharides can react with the amino groups of proteins to form Schiff bases, which rearrange to form a more stable amino ketone linkage. Such nonspecifically modified proteins often do not function effectively. Glucose has a strong tendency to exist in the ring formation and, consequently, relatively little tendency to modify proteins.

Q.2- Justify the statement- ‘Hexokinase Traps Glucose in the Cell and begins Glycolysis’.

Answer- Glucose enters cells through specific transport proteins and has one principal fate: it is phosphorylated by ATP to form glucose 6-phosphate. The transfer of the phosphoryl group from ATP to the hydroxyl group on carbon 6 of glucose is catalyzed by Hexokinase (Figure-1).

 

 

 

 

 

 

 

 

Figure-1 showing the phosphorylation of Glucose to Glucose-6- Phosphate catalyzed by Hexokinase

The incorporation of a phosphate into glucose in this energetically favourable reaction is important for several reasons. First, phosphorylation keeps the substrate in the cell. Glucose is a neutral molecule and could diffuse across the cell membrane, but phosphorylation confers a negative charge on glucose, and the plasma membrane is essentially impermeable to glucose-6-phosphate (Figure-2). Moreover, rapid conversion of glucose to glucose-6-phosphate keeps the intracellular concentration of glucose low, favoring diffusion of glucose into the cell. In addition the addition of the phosphoryl group begins to destabilize glucose, thus facilitating its further metabolism.

Further more, because regulatory control can be imposed only on reactions not at equilibrium, the favorable thermodynamics of this first reaction makes it an important site for regulation.

 

 

 

 

 

 

 

 

Figure-2- Phosphorylation of glucose to glucose-6-phosphate by ATP creates a charged molecule that cannot easily cross the plasma membrane.

 

Q.3- What are the important differences between Hexokinase and Glucokinase?

 Answer-

Hexokinase -In most animal, plant, and microbial cells, the enzyme that phosphorylates glucose is hexokinase. Magnesium ion (Mg2+) is required for this reaction, as for the other kinase enzymes in the glycolytic pathway. The true substrate for the hexokinase reaction is MgATP2-. The apparent Km for glucose of the animal skeletal muscle enzyme is approximately 0.05 mM/L, and the enzyme thus operates efficiently at normal blood glucose levels of 4 mM or so. Different body tissues possess different isozymes of hexokinase, each exhibiting somewhat different kinetic properties.

The animal enzyme is allosterically inhibited by the product, glucose-6-phosphate. High levels of glucose-6-phosphate inhibit hexokinase activity until consumption by glycolysis lowers its concentration. The hexokinase reaction is one of three points in the glycolysis pathway that are regulated. This is a very important regulatory step, since it prevents the consumption of too much cellular ATP to form G6P when glucose is not limiting. Hexokinase is, therefore, well-adapted as the initiator of glucose metabolism in tissues utilizing glucose as an energy source, but not as the initiator of energy storage in the liver(Figure-3).

As the generic name implies, hexokinase can phosphorylate a variety of hexose sugars, including glucose, mannose, and fructose. However, its affinity for these sugars varies greatly dependent upon their structures.  Hexokinase reacts strongly with glucose, while its affinity for fructose and galactose is relatively low.  Furthermore, glucose is a potent competitive inhibitor of the binding of galactose and fructose to hexokinase.  This excludes active handling of fructose and galactose by hexokinase at the concentrations found in our bodies. 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-3 -showing the entry of glucose in the cell and subsequent  phosphorylation by Hexokinase/Glucokinase. Hexokinase activity is inhibited by product, while Glucokinase activity handles glucose load.

Glucokinase occurs in cells in the liver, pancreas, gut, and brain of humans and most other vertebrates. In each of these organs it plays an important role in the regulation of carbohydrate metabolism by acting as a glucose sensor, triggering shifts in metabolism or cell function in response to rising or falling levels of glucose, such as occur after a meal or when fasting. Mutations of the gene for this enzyme can cause unusual forms of diabetes or hypoglycemia.

It requires a much higher glucose concentration for maximal activity. It is thus most active when glucose is very high in the portal vein, immediately after consumption of a carbohydrate-rich meal. The Km of the liver enzyme,around 5-6 mmol/L, lies above fasting blood glucose levels.  This means that Glucokinase activity is “turned on ” by the glucose in portal blood following a meal (10-30 mmolar), and it must be “turned off” after glucose from the meal is absorbed.

It has a high Vmax, allowing the liver to effectively remove excess glucose, and minimize hyperglycemia after eating. Glucokinase is not inhibited by G6P. Glucokinase is an inducible enzyme—the amount present in the liver is controlled by insulin (secreted by the pancreas). (Patients with diabetes mellitus produce insufficient insulin. They have low levels of Glucokinase, cannot tolerate high levels of blood glucose, and produce little liver glycogen.)

                                         Hexokinase                                    Glucokinase

Tissue distribution: Most tissues                                 Liver and β cells of Pancreas

Km                                 Low (0.05 mM)                             High (10 mM)

Vmax                            Low                                                     High

Inhibition by G6P    Yes                                                      No

Inducible                     No                                                       Yes

In the liver, the action of Glucokinase is opposed by the action of glucose-6-phosphatase. The balance between glucokinase and glucose-6-phosphatase slides back and forth, increasing uptake to the liver and phosphorylation when the level of blood glucose is high, and releasing glucose from G-6-P  when blood glucose falls. 

Glucokinase activity can be rapidly amplified or damped in response to changes in the glucose supply, typically in fed and fasting states. Regulation occurs at several levels and speeds, and is influenced by many factors which mainly affect two general mechanisms:

  1. Glucokinase activity can be amplified or reduced in minutes by actions of the Glucokinase regulatory protein (GKRP). The actions of this protein are influenced by small molecules such as glucose and fructose.
  2. The amount of Glucokinase can be increased by synthesis of new protein. Insulin is the principal signal for increased transcription (Induction), operating mainly by way of a transcription factor called sterol regulatory element binding protein-1c (SREBP1c). This occurs within an hour after a rise in insulin levels, as after a carbohydrate meal.

 

Q.4- Give a brief account of glycolysis mentioning the steps and the energy yield per molecule of glucose.

Answer- Glycolysis is the stepwise degradation of glucose (and other simple sugars). Glycolysis is a paradigm of metabolic pathways. Carried out in the cytosol of cells, it is unique, in that it can function either aerobically or anaerobically, depending on the availability of oxygen and the electron transport chain. Erythrocytes, which lack mitochondria, are completely reliant on glucose as their metabolic fuel, and metabolize it by anaerobic glycolysis. However, to oxidize glucose beyond pyruvate (the end product of glycolysis) requires both oxygen and mitochondrial enzyme systems such as the pyruvate dehydrogenase complex, the citric acid cycle, and the respiratory chain.

Living things first appeared in an environment lacking O2, and glycolysis was an early and important pathway for extracting energy from nutrient molecules. It played a central role in anaerobic metabolic processes during the first 2 billion years of biological evolution on earth. Modern organisms still employ glycolysis to provide precursor molecules for aerobic catabolic pathways (such as the tricarboxylic acid cycle) and as a short-term energy source when oxygen is limiting.

Overview of Glycolysis

Glycolysis consists of two phases. In the first, a series of five reactions, glucose is broken down to two molecules of glyceraldehyde-3-phosphate. In the second phase, five subsequent reactions convert these two molecules of glyceraldehyde-3-phosphate into two molecules of pyruvate. Phase 1 consumes two molecules of ATP. The later stages of glycolysis result in the production of four molecules of ATP. The net is 4 – 2 = 2 molecules of ATP produced per molecule of glucose.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-4- showing  an overview of Glycolysis

Reactions of Glycolysis

Most of the details of this pathway (the first metabolic pathway to be elucidated) were worked out in the first half of the 20th century by the German biochemists Otto Warburg, G. Embden, and O. Meyerhof. In fact, the sequence of reactions in is often referred to as the Embden-Meyerhof pathway.

The First Phase of Glycolysis

One way to synthesize ATP using the metabolic free energy contained in the glucose molecule would be to convert glucose into one (or more) of the high-energy phosphates that have standard-state free energies of hydrolysis more negative than that of ATP. In fact, in the first stage of glycolysis, glucose is converted into two molecules of glyceraldehyde-3-phosphate. Energy released from this high-energy molecule in the second phase of glycolysis is then used to synthesize ATP.

Reaction 1: Phosphorylation of Glucose by Hexokinase or Glucokinase —The First Priming Reaction

Glucose enters glycolysis by phosphorylation to glucose 6-phosphate, catalyzed by hexokinase, using ATP as the phosphate donor. Under physiologic conditions, the phosphorylation of glucose to glucose 6-phosphate can be regarded as irreversible. (Reaction -1) Hexokinase is inhibited allosterically by its product, glucose 6-phosphate.

In tissues other than the liver (and pancreatic beta-islet cells), the availability of glucose for glycolysis (or glycogen synthesis in muscle and lipogenesis in adipose tissue) is controlled by transport into the cell, which in turn is regulated by insulin. Hexokinase has a high affinity (low Km) for glucose, and in the liver it is saturated under normal conditions, and so acts at a constant rate to provide glucose 6-phosphate to meet the cell’s need. Liver cells also contain an isoenzyme of hexokinase, Glucokinase, which has a Km very much higher than the normal intracellular concentration of glucose. The function of glucokinase in the liver is to remove glucose from the blood following a meal, providing glucose 6-phosphate in excess of requirements for glycolysis, which is used for glycogen synthesis and lipogenesis(Figure-3).

The formation of such a phosphoester is thermodynamically unfavorable and requires energy input to operate in the forward direction .The energy comes from ATP, a requirement that at first seems counterproductive. Glycolysis is designed to make ATP, not consume it. However, the hexokinase, glucokinase reaction is one of two priming reactions in the cycle.

Glucose 6-phosphate is an important compound at the junction of several metabolic pathways: glycolysis, gluconeogenesis, the pentose phosphate pathway, Glycogenesis, and glycogenolysis (Figure-19).

Reaction 2: Phosphoglucoisomerase Catalyzes the Isomerization of Glucose-6-Phosphate

The second step in glycolysis is a common type of metabolic reaction: the isomerization of a sugar. In this particular case, the carbonyl oxygen of glucose-6-phosphate is shifted from C-1 to C-2. This amounts to isomerization of an aldose (glucose-6-phosphate) to a ketose—fructose-6-phosphate (Figure-5). The reaction is necessary for two reasons. First, the next step in glycolysis is phosphorylation at C-1, and the hemiacetal -OH of glucose would be more difficult to phosphorylate than a simple primary hydroxyl. Second, the isomerization to fructose (with a carbonyl group at position 2 in the linear form) activates carbon C-3 for cleavage in the fourth step of glycolysis. The enzyme responsible for this isomerization is Phosphoglucoisomerase, also known as glucose phosphate Isomerase. In humans, the enzyme requires Mg2+ for activity and is highly specific for glucose-6-phosphate.

Reaction 3: Phospho fructokinase —The Second Priming Reaction

The action of Phosphoglucoisomerase, “moving” the carbonyl group from C-1 to C-2, creates a new primary alcohol function at C-1 (see Figure-5). The next step in the glycolytic pathway is the phosphorylation of this group by phosphofructo kinase. Once again, the substrate that provides the phosphoryl group is ATP. Just as the hexokinase reaction commits the cell to taking up glucose, the phosphofructo kinase reaction commits the cell to metabolizing glucose rather than converting it to another sugar or storing it. Similarly, just as the large free energy change of the hexokinase reaction makes it a likely candidate for regulation, so the phosphofructo kinase reaction is an important site of regulation—indeed, the most important site in the glycolytic pathway.

Reaction 4: Cleavage of Fructose-1,6-bis P by Fructose Bisphosphate Aldolase

Fructose bisphosphate aldolase cleaves fructose-1,6-bisphosphate between the C-3 and C-4 carbons to yield two triose phosphates. The products are Dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate.

Reaction 5: Triose Phosphate Isomerase

Of the two products of the Aldolase reaction, only glyceraldehyde-3-phosphate goes directly into the second phase of glycolysis. The other triose phosphate, Dihydroxyacetone phosphate, must be converted to glyceraldehyde-3-phosphate by the enzyme triose phosphate Isomerase.

This reaction thus permits both products of the aldolase reaction to continue in the glycolytic pathway, and in essence makes the C-1, C-2, and C-3 carbons of the starting glucose molecule equivalent to the C-6, C-5, and C-4 carbons, respectively.  The triose phosphate Isomerase reaction completes the first phase of glycolysis, each glucose molecule that passes through being converted to two molecules of glyceraldehyde-3-phosphate.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-5- Showing reactions of Glycolysis

The Second Phase of Glycolysis

The second half of the glycolytic pathway involves the reactions that convert the metabolic energy in the glucose molecule into ATP.

Reaction 6: Glyceraldehyde-3-Phosphate Dehydrogenase

In the first glycolytic reaction to involve oxidation-reduction, glyceraldehyde-3-phosphate is oxidized to 1,3-bisphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase.

The enzyme catalyzing this oxidation, glyceraldehyde 3-phosphate dehydrogenase, is NAD-dependent. Structurally, it consists of four identical polypeptides (monomers) forming a tetramer. Four —SH groups are present on each polypeptide, derived from cysteine residues within the polypeptide chain. One of the —SH groups is found at the active site of the enzyme .The substrate initially combines with this —SH group, forming a thio hemiacetal that is oxidized to a thiol ester; the hydrogens removed in this oxidation are transferred to NAD+. The enzyme can be inactivated by reaction with iodoacetate, which reacts with and blocks the essential cysteine sulfhydryl.

The glyceraldehyde-3-phosphate dehydrogenase reaction is the site of action of arsenate (AsO43-),(Figure-6), an anion analogous to phosphate. Arsenate is an effective substrate in this reaction, forming 1-arseno-3-phosphoglycerate, but acyl arsenates are quite unstable and are rapidly hydrolyzed. 1-Arseno-3-phosphoglycerate breaks down to yield 3-phosphoglycerate, the product of the seventh reaction of glycolysis. The result is that glycolysis continues in the presence of arsenate, but the molecule of ATP formed in reaction 7 ( phosphoglycerate kinase ) is not made because this step has been bypassed. The lability of 1-arseno-3-phosphoglycerate effectively uncouples the oxidation and phosphorylation events, which are normally tightly coupled in the glyceraldehyde-3-phosphate dehydrogenase reaction.

 

 

 

 

 

 

 

 

 

 

Figure-6- showing the structure of 1- Arseno-3-phosphoglycerate

 Reaction 7 : Phosphoglycerate Kinase

The enzyme phosphoglycerate kinase transfers a phosphoryl group from 1,3-bisphosphoglycerate to ADP to form an ATP (Figure-7). Because each glucose molecule sends two molecules of glyceraldehyde-3-phosphate into the second phase of glycolysis and because two ATPs were consumed per glucose in the first-phase reactions, the phosphoglycerate kinase reaction “pays off” the ATP debt created by the priming reactions. As might be expected for a phosphoryl transfer enzyme, Mg2+ ion is required for activity, and the true nucleotide substrate for the reaction is MgADP-. It is appropriate to view the sixth and seventh reactions of glycolysis as a coupled pair, with 1,3-bisphosphoglycerate as an intermediate.

ADP has been phosphorylated to form ATP at the expense of a substrate, namely, glyceraldehyde-3-phosphate. This is an example of substrate-level phosphorylation. The other kind of phosphorylation, oxidative phosphorylation, is driven energetically by the transport of electrons from appropriate coenzymes and substrates to oxygen.

An important regulatory molecule, 2,3-bisphosphoglycerate, is synthesized and metabolized by a pair of reactions that make a detour around the phosphoglycerate kinase reaction. 2,3-BPG, which stabilizes the deoxy form of hemoglobin and is primarily responsible for the cooperative nature of oxygen binding by hemoglobin, is formed from 1,3-bisphosphoglycerate by bisphosphoglycerate mutase (Figure-5).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure -7- Formation and decomposition of 2,3-bisphosphoglycerate.

Hydrolysis of 2,3-BPG is carried out by 2,3-bisphosphoglycerate phosphatase . Although other cells contain only a trace of 2,3-BPG, erythrocytes typically contain 4 to 5 mM 2,3-BPG.

Reaction 8: Phosphoglycerate Mutase

The remaining steps in the glycolytic pathway prepare for synthesis of the second ATP equivalent. This begins with the phosphoglycerate mutase reaction (Figure-7), in which the phosphoryl group of 3-phosphoglycerate is moved from C-3 to C-2. (The term mutase is applied to enzymes that catalyze migration of a functional group within a substrate molecule.)

Reaction 9: Enolase

This reaction of glycolysis makes a high-energy phosphate in preparation for ATP synthesis.

Enolase catalyzes the formation of phosphoenolpyruvate from 2-phosphoglycerate (Figure-5). The reaction in essence involves a dehydration—the removal of a water molecule—to form the enol structure of PEP. The enzyme is strongly inhibited by fluoride ion in the presence of phosphate. Inhibition arises from the formation of fluorophosphate (FPO32-), which forms a complex with Mg2+ at the active site of the enzyme. 

Reaction 10: Pyruvate Kinase

The second ATP-synthesizing reaction of glycolysis is catalyzed by pyruvate kinase, which brings the pathway at last to its pyruvate branch point. Pyruvate kinase mediates the transfer of a phosphoryl group from phosphoenolpyruvate to ADP to make ATP and pyruvate (Figure5 and 18). The reaction requires Mg2+ ion and is stimulated by K+ and certain other monovalent cations.

For each glucose molecule in the glycolysis pathway, two ATPs are made at the pyruvate kinase stage (because two triose molecules were produced per glucose in the aldolase reaction). Because the pathway broke even in terms of ATP at the phosphoglycerate kinase reaction (two ATPs consumed and two ATPs produced), the two ATPs produced by pyruvate kinase represent the “payoff” of glycolysis —a net yield of two ATP molecules.

The Metabolic Fates of NADH and Pyruvate —The Products of Glycolysis

In addition to ATP, the products of glycolysis are NADH and pyruvate. Their processing depends upon other cellular pathways. NADH must be recycled to NAD+, lest NAD+ become limiting in glycolysis. NADH can be recycled by both aerobic and anaerobic paths, either of which results in further metabolism of pyruvate. What a given cell does with the pyruvate produced in glycolysis depends in part on the availability of oxygen.

Under aerobic conditions, pyruvate can be sent into the citric acid cycle, where it is oxidized to CO2 with the production of additional NADH (and FADH2). Under aerobic conditions, the NADH produced in glycolysis and the citric acid cycle is reoxidized to NAD+ in the mitochondrial electron transport chain.

Under anaerobic conditions, the NADH cannot be reoxidized through the respiratory chain to oxygen. Pyruvate is reduced by the NADH to lactate, catalyzed by lactate dehydrogenase. There are different tissue specific isoenzymes lactate dehydrogenases that have clinical significance. The reoxidation of NADH via lactate formation allows glycolysis to proceed in the absence of oxygen by regenerating sufficient NAD+ for another cycle of the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase (figure-8)

 

 

 

 

 

 

 

 

 

 

Figure–8 showing coupling of reactions

Tissues That Function under Hypoxic Conditions Produce Lactate

This is true of skeletal muscle, particularly the white fibers, where the rate of work output, and hence the need for ATP formation, may exceed the rate at which oxygen can be taken up and utilized. Glycolysis in erythrocytes always terminates in lactate, because the subsequent reactions of pyruvate oxidation are mitochondrial, and erythrocytes lack mitochondria. Other tissues that normally derive much of their energy from glycolysis and produce lactate include brain, gastrointestinal tract, renal medulla, retina, and skin. The liver, kidneys, and heart usually take up lactate and oxidize it but will produce it under hypoxic conditions.

Energy yield per molecule of Glucose oxidized through Glycolysis

The net reaction in the transformation of glucose into pyruvate is:


 

 

Under anaerobic conditions Electron transport chain does not operate so the ATP is only formed by substrate level phosphorylation. Hence the total energy yield through glycolysis in the absence of oxygen is only 2 ATP per Mol of Glucose.

 

 

 

 

 

 

 

 

 

 

 

 

 

Q.5- Discuss the regulation of Glycolysis. Why is phosphofructokinase rather than hexokinase the pacemaker of glycolysis?

Answer- The flux through the glycolytic pathway must be adjusted in response to conditions both inside and outside the cell. The rate of conversion of glucose into pyruvate is regulated to meet two major cellular needs: (1) the production of ATP, generated by the degradation of glucose, and (2) the provision of building blocks for synthetic reactions, such as the formation of fatty acids.

Flux through a metabolic pathway can be regulated in several ways:

1. Availability of substrate

2. Concentration of enzymes responsible for rate-limiting steps

3. Allosteric regulation of enzymes

4. Covalent modification of enzymes (e.g. phosphorylation)

Generally, enzymes that catalyze essentially irreversible steps in metabolic pathways are potential sites for regulatory control.  Although most of the reactions of glycolysis are reversible, three are markedly exergonic and must therefore be considered physiologically irreversible. The enzymes responsible for catalyzing these three steps, hexokinase (or glucokinase) for step 1, phosphofructo kinase for step 3, and pyruvate kinase for step 10, are the primary steps for allosteric enzyme regulation.

Availability of substrate (in this case, glucose), is another general point for regulation.

The concentration of these three enzymes in the cell is regulated by hormones that affect their rates of transcription. Insulin upregulates the transcription of Glucokinase, phosphofructo kinase,  and pyruvate kinase, while glucagon down regulates their transcription. These effects take place over a period of hours to days, and generally reflect whether a person is well-fed or starving.

1) Regulation at the level of Hexokinase and Glucokinase-

The Hexokinase enzyme is allosterically inhibited by the product, glucose-6-phosphate. Glucokinase is highly specific for D-glucose, has a much higher Km for glucose (approximately 10.0 mM ), and is not product-inhibited. With such a high Km for glucose, Glucokinase becomes important metabolically only when liver glucose levels are high. Glucokinase is an inducible enzyme—the amount present in the liver is controlled by insulin. The low glucose affinity of Glucokinase in the liver gives the brain and muscles first call on glucose when its supply is limited, whereas it ensures that glucose will not be wasted when it is abundant.

2) Regulation of Phospho fructokinase

Phospho fructokinase is the “valve” controlling the rate of glycolysis.

a) Role of ATP

 ATP is an allosteric inhibitor of this enzyme.

In the presence of high ATP concentrations, the Km for fructose-6-phosphate is increased, glycolysis thus “turns off.” ATP elicits this effect by binding to a specific regulatory site that is distinct from the catalytic site. AMP reverses the inhibitory action of ATP, and so the activity of the enzyme increases when the ATP/AMP ratio is lowered. In other words, glycolysis is stimulated as the energy charge falls. A fall in pH also inhibits Phosphofructokinase activity. The inhibition of phosphofructokinase by H+ prevents excessive formation of lactic acid and a precipitous drop in blood pH (acidosis).

b) Role of Citrate

 Glycolysis also furnishes carbon skeletons for biosyntheses, and so a signal indicating whether building blocks are abundant or scarce should also regulate phosphofructokinase.

Indeed, phosphofructokinase is inhibited by citrate, an early intermediate in the citric acid cycle. A high level of citrate means that biosynthetic precursors are abundant and additional glucose should not be degraded for this purpose. Citrate inhibits phosphofructokinase by enhancing the inhibitory effect of ATP. Inhibition of glycolysis by citrate ensures that glucose will not be committed to these activities if the citric acid cycle is already saturated.

c) Role of Fr 2,6 bisphosphate

Phosphofructokinase is also regulated by D-fructose-2,6-bisphosphate, a potent allosteric activator that increases the affinity of phosphofructokinase for the substrate fructose-6-phosphate. Stimulation of phosphofructokinase is also achieved by decreasing the inhibitory effects of ATP. Fructose-2,6-bisphosphate increases the net flow of glucose through glycolysis by stimulating phosphofructokinase and, by inhibiting fructose-1,6-bisphosphatase, the enzyme that catalyzes this reaction in the opposite direction.

 Why is phosphofructokinase rather than hexokinase the pacemaker of glycolysis? The reason becomes evident on noting that glucose 6-phosphate is not solely a glycolytic intermediate. Glucose 6-phosphate can also be converted into glycogen or it can be oxidized by the pentose phosphate pathway to form NADPH. The first irreversible reaction unique to the glycolytic pathway, the committed step,is the phosphorylation of fructose 6- phosphate to fructose 1,6-bisphosphate. Thus, it is highly appropriate for phosphofructokinase to be the primary control site in glycolysis. In general, the enzyme catalyzing the committed step in a metabolic sequence is the most important control element in the pathway.

3) Regulation of pyruvate Kinase

Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine.

Furthermore, liver pyruvate kinase is regulated by covalent modification. Hormones such as glucagon activate a cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme. The phosphorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher Km for PEP, so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the gluconeogenesis pathway, instead of going on through glycolysis and the citric acid cycle (or fermentation routes). This hormone-triggered phosphorylation, prevents the liver from consuming glucose when it is more urgently needed by brain and muscles.

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure –11- showing the regulation of pyruvate kinase by allosteric effectors and by covalent modification

 Q.6 – Explain the effect of increasing the concentration of each of the following metabolites on the net rate of glycolysis: (a) glucose-6-phosphate (b) fructose-1,6-bisphosphate (c) citrate.

Answer- The following effects are seen –

(a)     Glucose-6-phosphate- It is a product of hexokinase catalyzed first reaction of glycolysis. The Hexokinase enzyme is allosterically inhibited by the product, glucose-6-phosphate. So glycolysis will be inhibited.

(b)     fructose-1,6-bisphosphate- It is a product of Phosphofructokinase catalyzed reaction and is a positive modifier of Pyruvate kinase enzyme. The rate of glycolysis will increase. 

(c)      Citrate- It is a negative allosteric modifier of Phosphofructokinase enzyme. So the rate of glycolysis will decrease.

 

Q.7- Discuss the formation and degradation of Fructose 2,6 bisphosphate. What is feed forward stimulation  in glycolysis?

Or

Discuss the role of Fr 2,6 bisphosphate in the regulation of glycolysis.

Or

How is the concentration of fructose 2,6-bisphosphate appropriately controlled?

Answer-

Fr 2, 6 bisphosphate is an important regulator of glycolysis. It is a positive modifier of Phosphofructokinase -1 enzyme.

 Two enzymes regulate its concentration by phosphorylating fructose 6-phosphate and dephosphorylating fructose 2,6- bisphosphate.

Synthesis of fructose2,6-bisphosphate

Fructose 2,6-bisphosphate is formed in a reaction catalyzed by phosphofructokinase 2 (PFK2), a different enzyme from phosphofructokinase.

Degradation of Fructose 2,6-bisphosphate

 Fructose 2,6-bisphosphate is hydrolyzed to fructose 6-phosphate by a specific phosphatase, fructose bisphosphatase 2 (FBPase2).

Regulation of concentration of Fructose 2,6-bisphosphate

 The striking finding is that both PFK2 and FBPase2 are present in a single 55-kd polypeptide chain. This bifunctional enzyme contains an N-terminal regulatory domain, followed by a kinase domain and a phosphatase domain. The bifunctional enzyme itself probably arose by the fusion of genes encoding the kinase and phosphatase domains.

 

 

 

 

 

 

 

 

 

 

Figure –12- showing the orientation of functional domains of bifunctional enzyme

 In the liver, the concentration of fructose 6-phosphate rises when blood-glucose concentration is high, and the abundance of fructose 6-phosphate accelerates the synthesis of F- 2,6-BP (Figure-13). Hence, an abundance of fructose 6-phosphate leads to a higher concentration of F-2,6-BP, which in turn stimulates phosphofructokinase. The product of PFK-1 catalyzed reaction Fr 1,6 bisphosphate further stimulates pyruvate kinase enzyme. Such a process is called feed forward stimulation.

The activities of PFK2 and FBPase2 are reciprocally controlled by phosphorylation of a single serine residue. When glucose is scarce, a rise in the blood level of the hormone glucagon triggers a cyclic AMP cascade, leading to the phosphorylation of this bifunctional enzyme by protein kinase A. This covalent modification activates FBPase2 and inhibits PFK2, lowering the level of F-2,6-BP. Thus, glucose metabolism by the liver is curtailed(figure-13).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure- 13-showing the role of fructose 2,6 bisphosphate in the regulation of PFK-1 enzyme of Glycolysis

Conversely, when glucose is abundant, the enzyme loses its attached phosphate group. This covalent modification activates PFK2 and inhibits FBPase2, raising the level of F-2,6-BP and accelerating glycolysis.

Thus, when glucose is abundant as during fed state, glycolysis is stimulated and when glucose is limiting as during fasting or starvation glycolysis is inhibited.  These effects are brought about by hormones affecting the concentration of fr 2,6 bisphosphate through action on the bifunctional enzymes fr 2,6 bisphosphatase and PFK-2.

Q.8- Red blood cells have an alternate pathway for glycolysis that produces an intermediate that is essential for the function of the red blood cell. This detour bypasses an ATP generating step. Discuss this detour in terms of the intermediate that is generated and the function of red blood cells.

Or

What is Luebering-Rapapport pathway? What is its significance?

Answer- 2,3-BPG, is  the most concentrated organophosphate in the erythrocytes.  It is synthesized from Glucose by Luebering-Rapapport pathway which is a diversion from the main glycolytic pathway.

Luebering-Rapapport pathway

In erythrocytes, the reaction catalyzed by phosphoglycerate kinase may be bypassed, to some extent by the reaction of bisphosphoglycerate mutase, which catalyzes the conversion of 1,3-bisphosphoglycerate to 2,3-bisphosphoglycerate, followed by hydrolysis to 3-phosphoglycerate and Pi, catalyzed by 2,3-bisphosphoglycerate phosphatase (Figure-14). This alternative pathway involves no net yield of ATP from glycolysis. However, it does serve to provide 2,3-bisphosphoglycerate, which binds to hemoglobin, decreasing its affinity for oxygen, and so making oxygen more readily available to tissues .


 

 

 

 

Figure-14 -showing the formation of 2,3 BPG

There is a delicate balance between the need to generate ATP to support energy requirements for cell metabolism and the need to maintain appropriate oxygenation/deoxygenation status of hemoglobin. This balance is maintained by dephosphorylation of 1,3-BPG to 2,3-BPG, which enhances the deoxygenation of hemoglobin. Low pH inhibits the activity of bisphosphoglyceromutase and activates bisphosphoglycerate phosphatase, which favors generation of ATP.

 Significance of 2,3-bisphosphoglycerate

a) Unloading of Oxygen-

When 2,3-BPG binds to deoxyhemoglobin, it acts to stabilize the low oxygen affinity state (T state) of the oxygen carrier,  exploiting the molecular symmetry and positive polarity by forming salt bridges with lysine and histidine residues in the four subunits of hemoglobin.

The R state, with oxygen bound to a heme group, has a different conformation and does not allow this interaction. By selectively binding to deoxyhemoglobin, 2,3-BPG stabilizes the T state conformation, making it harder for oxygen to bind hemoglobin and more likely to be released to adjacent tissues.(Figure 15)

b) Effect of Hypoxia-

 2,3-BPG can help to  prevent tissue hypoxia in conditions where it is most likely to occur. Conditions of low tissue oxygen concentration such as high altitude (2,3-BPG levels are higher in those acclimated to high altitudes), airway obstruction,  anemias or congestive heart failure will tend to cause RBCs to generate more 2,3-BPG in their effort to generate energy by allowing more oxygen to be released in tissues deprived of oxygen.

This release is potentiated by the Bohr effect in tissues with high energetic demands.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure- 15-showing the effect of Binding of 2,3 BPG to hemoglobin

c) Smoking and 2,3 BPG

Cigarette smoking has been shown to increase both respiratory and blood CO levels.  CO has a much greater affinity for hemoglobin than does O2. It will bind to hemoglobin and prevent O2 from binding to hemoglobin.  As a way to adapt to this problem caused by smoking, similar to the body at high altitudes, 2,3-BPG concentrations increase in the red blood cells  to release more  O2 to the tissues.

d) Fetal hemoglobin (HbF) and 2,3 BPG

Fetal hemoglobin (HbF) exhibits a low affinity for 2,3-BPG, resulting in a higher binding affinity for oxygen. This increased oxygen-binding affinity relative to that of adult hemoglobin (HbA) is due to HbF’s having two α/γ dimers as opposed to the two α/β dimers of HbA. The positive histidine residues of HbA β-subunits that are essential for forming the 2,3-BPG binding pocket are replaced by serine residues in HbF γ-subunits. Like that, histidine nº143 gets lost, so 2,3-BPG has difficulties in linking to the fetal hemoglobin, so the affinity of fetal hemoglobin for O2 increases . That’s the way O2 flows from the mother to the fetus.

 

Q.9 – Explain which metabolic intermediate(s) will accumulate when each of the following is added to cell-free extracts capable of glycolysis: (a) fluoride, which inhibits Enolase (b) an inhibitor of lactate dehydrogenase (c) an inhibitor of pyruvate kinase.

Answer- The following effects would be seen in response to the presence of a specific inhibitor-

(a) fluoride- Fluoride acts primarily by inhibiting enolase in the glycolytic pathway, which catalyzes the conversion of 2, phosphoglycerate to phosphoenol pyruvate (Figure-16). Fluoride strongly inhibits the enzyme in the presence of inorganic phosphate. The inhibitory species is the fluorophosphate ion, which when bound to magnesium forms a complex with enolase and inactivates the enzyme.

 In the presence of fluoride 2, phosphoglycerate will first increase in concentration and subsequently all the intermediates above the block will accumulate causing inhibition of glycolysis.  A mixture of sodium fluoride and potassium oxalate is used while collecting the blood sample for glucose estimation. Sodium fluoride prevents glucose loss from the sample by preventing glycolysis.

 

 

 

 

 

 

 

 

Figure-16- showing the Enolase catalyzed reaction

 

(b) An inhibitor of lactate dehydrogenase- Oxamate is a competitive inhibitor of lactate dehydrogenase enzyme. Lactate dehydrogenase catalyzes the conversion of pyruvate to Lactate (figure-17). It is a reversible reaction. Lactate is the end product of glycolysis in the absence of oxygen as well as in those cells which lack mitochondria. This reaction is coupled with glyceraldehyde-3 phosphate dehydrogenase reaction for the regeneration of NAD+ for the continuation of glycolysis. In the presence of Oxamate there will be accumulation of pyruvate and of  the other intermediates proximal to the block,  resulting in inhibition of glycolysis.

 

 

 

 

 

 

Figure-17-showing conversion of pyruvate to lactate catalysed by Lactate dehydrogenase

Tumors have a much greater dependence than normal tissues on anaerobic glycolysis for energy generation. Inhibition of anaerobic glycolysis by selective LDH inhibitors might be used to obtain a significant therapeutic gain in combination treatments with cytotoxic drugs or radiotherapy in cancers.

(c) An inhibitor of pyruvate kinase-

Pyruvate kinase catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, yielding one molecule of pyruvate and one molecule of ATP(Figure-18).

 

 

 

 

 

 

 

Figure-18- showing the formation of pyruvate from Phosphoenol pyruvate

In the presence of an inhibitor of pyruvate kinase, Phosphoenol pyruvate will accumulate, glycolysis will shut down. Under physiological conditions pyruvate kinase is inhibited either in the presence of allosteric inhibitors or by phosphorylation, under these conditions PEP is used for the formation of Glucose by the process of gluconeogenesis.

 

Q.10- Glucose-6-phosphate is at the crossroads of 3 metabolic pathways in liver cells. Name the pathways and discuss the metabolic conditions that determine which pathway will prevail.

Answer- Glucose-6-phosphate is common to several metabolic pathways. It occupies a branch point in glucose metabolism. The fate of Glucose-6 –phosphate depends on the type and the need of the cell (Figure-19)-

 

 

 

 

 

 

 

 

Figure-19- showing the fate of glucose-6-phosphate

1)      Glycolysis- Glucose-6 –phosphate may get converted to fructose- 6 phosphate for the continuation of glycolysis if the cell is in low energy state. Almost all the cells of the body are capable of oxidizing glucose by Glycolysis

2)      Glycogenesis and Uronic acid pathway- Glucose-6 –phosphate may get converted to Glucose-1-phosphate to be subsequently converted to Glycogen.  This reaction takes place in conditions of glucose excess and the extra glucose is stored as glycogen.  Glycogen is synthesized mainly in liver and muscles.Glucose-1-phosphate produced from Glucose-6 –phosphate may enter Uronic acid pathway for the production of Glucuronic acid which is required for the detoxification of xenobiotics. This pathway mainly takes place in liver.

3)      HMP pathway -Glucose-6 –phosphate may enter the HMP pathway for the synthesis of NADPH and pentoses. NADPH is required for the reductive biosynthesis while pentoses are required for the synthesis of coenzymes and nucleotides. This pathway operates excessively in those cells which are actively involved in reductive biosynthesis like liver, adrenal cortex, placenta, lactating mammary glands etc.

4)      Glycogenolysis and Gluconeogenesis- Glucose-6-P may be converted back to glucose by the enzyme Glucose-6-phosphatase. This is important during glycogenolysis and gluconeogenesis.

5)      Fructose- 6-P produced from Glucose-6 –phosphate may be utilized for the production of Glucosamines.

Q.11- Justify the statement –“Muscle cells derive almost all of the ATP consumed during exertion from anaerobic glycolysis.”

Or

Why is lactate, rather than pyruvate, produced by normal muscle when it is working anaerobically?

Answer-

Under anaerobic conditions, pyruvate is converted to lactate by the enzyme lactate dehydrogenase (LDH), Pyruvate reduction occurs in tissues that normally experience minimal access to blood flow (e.g., the cornea of the eye) and also in rapidly contracting skeletal muscle. When skeletal muscles are exercised strenuously, the available tissue oxygen is consumed, and the pyruvate generated by glycolysis can no longer be oxidized in the TCA cycle. Instead, excess pyruvate is reduced to lactate by lactate dehydrogenase. The conversion of pyruvate to lactate, under anaerobic conditions, provides the cell with a mechanism for the oxidation of NADH (produced during the G3PDH reaction) to NAD+ which occurs during the LDH catalyzed reaction. This reduction is required since NAD+ is a necessary substrate for G3PDH, without which glycolysis will cease. Normally, during aerobic glycolysis the electrons of cytoplasmic NADH are transferred to mitochondrial carriers of the oxidative phosphorylation pathway generating a continuous pool of cytoplasmic NAD+.

In anaerobic muscle tissue, lactate represents the end of glycolysis. Anyone who exercises to the point of consuming all available muscle oxygen stores knows the cramps and muscle fatigue associated with the buildup of lactic acid in the muscle. Most of this lactate must be carried out of the muscle by the blood and transported to the liver, where it can be resynthesized into glucose in gluconeogenesis.

Aerobic glycolysis generates substantially more ATP per mole of glucose oxidized than does anaerobic glycolysis. The utility of anaerobic glycolysis, to a muscle cell when it needs large amounts of energy, stems from the fact that the rate of ATP production from anaerobic glycolysis is approximately 100X faster than from oxidative phosphorylation. The requirement is to generate the maximum amount of ATP, for muscle contraction, in the shortest time frame. This is why muscle cells derive almost all of the ATP consumed during exertion from anaerobic glycolysis.

 

 Q.12 -Are substrates other than glucose oxidized in Glycolysis?

Answer- The glycolytic pathway begins with the breakdown of glucose, but other sugars simple or complex, if converted to any of the glycolytic intermediates can be used in the glycolytic pathway for the production of energy. The sugars like Fructose, Galactose. Mannose and even Glycerol produced from hydrolysis of triglycerides or obtained from diet or other sources can be oxidized through glycolysis.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-20- showing entry point of fructose and Galactose in to the pathway of glycolysis,

 

Q.13- What is the significance of glycolysis other than energy production?

Answer- Glycolysis is an important pathway for the production of energy especially under anaerobic conditions and in the cells lacking mitochondria, besides that the intermediates of glycolysis can be used for various purposes.

1)      Glucose-6-P is a common intermediate for a number of pathways and is used depending on the need of the cell, like glycogen synthesis, Uronic acid pathway, HMP pathway etc.

2)      Fructose-6-P is used for the synthesis of Glucosamines.

3)      Triose like glyceraldehyde-3-P and other glycolytic intermediates can be used   in the HMP pathway for the production of pentoses.

4)      Dihydroxy Acetone –phosphate can be used for the synthesis of Glycerol -3-P , which is used for the synthesis of Triglycerides or phospholipids.

5)      2,3 BPG is an important compound produced pathway in erythrocytes  in the glycolytic pathway for unloading of O2 to the peripheral tissues.

6)      The sugars like Fructose, Galactose. Mannose and even Glycerol can be oxidized in glycolysis.

7)      Out of the total 10 reactions of Glycolysis, 7 reactions are reversible and are used for the synthesis of Glucose by the process of Gluconeogenesis.

8)      Pyruvate the end product of glycolysis provides precursor for the TCA cycle and for the synthesis of other compounds.

 

Q.14- What is the cause of hemolytic anemia in patients suffering from Pyruvate kinase deficiency?

Answer- Pyruvate kinase lies at the end of the glycolytic pathway in RBCs followed only by lactate dehydrogenase. Pyruvate kinase activity is critical for the pathway and therefore critical for energy production. If ATP is not produced in amounts sufficient to meet the energy demand, then those functions are compromised. Energy is required to maintain the Na+/K+ balance within the RBC and to maintain the flexible discoid shape of the cell. In the absence of sufficient pyruvate kinase activity and therefore ATP, the ionic balance fails, and the membrane becomes misshapen. Cells reflecting pyruvate kinase insufficiency rather than a change in membrane composition are removed from the circulation by the macrophages of the spleen. This results in an increased number of circulating reticulocytes and possibly bone marrow hyperplasia, which is a biological response to lowered RBC count as a result of hemolysis of erythrocytes.

Enzyme defects that have been described include decreased substrate affinity, increased product inhibition, decreased response to activator, and thermal instability.

Important intermediates proximal to the PK defect influence erythrocyte function. Two- to 3-fold increases of 2, 3-bisphosphoglycerate levels result in a significant rightward shift in the hemoglobin-oxygen dissociation curve. Physiologically, the hemoglobin of affected individuals has an increased capacity to release oxygen into the tissues, thereby enhancing oxygen delivery. Thus, for a comparative hemoglobin and Haemtocrit level, an individual with PKD has an enhanced exercise capacity and fewer symptoms.

This disorder manifests clinically as a hemolytic anemia, but surprisingly, the symptomatology is less severe than hematological indices indicate. Presumably, this is due to enhanced oxygen delivery as a result of the defect. The clinical severity of this disorder varies widely, ranging from a mildly compensated anemia to severe anemia of childhood. Most affected individuals do not require treatment. Individuals who are most severely affected may die in utero of anemia or may require blood transfusions or Splenectomy, but most of the symptomatology is limited to early life and to times of physiologic stress or infection.

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Q.1- Give a brief account of digestion and absorption of dietary carbohydrates.

Answer- The digestion of carbohydrates involves hydrolysis to liberate oligosaccharides, disaccharides and finally monosaccharides.

Digestion of carbohydrates

Amylases Catalyze the Hydrolysis of Starch

The hydrolysis of starch is catalyzed by salivary and pancreatic amylases, which catalyze random hydrolysis of α (1-›4) glycoside bonds, yielding dextrins, then a mixture of glucose, maltose, and isomaltose (from the branch points in amylopectin).

The process of digestion starts in mouth by salivary alpha amylase, however due to shorter duration of stay of food in mouth, the digestion is left incomplete.

Gastric HCl causes hydrolysis of sucrose, while there is no hydrolytic enzyme present  in gastric juice for the digestion of carbohydrates.

Pancreatic amylase, an isoenzyme of salivary amylase, differs only in the optimum pH of action. Both the enzymes require Chloride ions for their action (Ion activated enzymes).(Figure-1)

Disaccharidases Are Brush Border Enzymes

The disaccharidases, maltase, sucrase-isomaltase (a bifunctional enzyme catalyzing hydrolysis of sucrose and isomaltose), lactase, and trehalase are located on the brush border of the intestinal mucosal cell where the resultant monosaccharides and others arising from the diet are absorbed. In most people, apart from those of northern European origin, lactase is gradually lost through adolescence, leading to lactose intolerance. Lactose remains in the intestinal lumen, where it is a substrate for bacterial fermentation to lactate, resulting in discomfort and diarrhea.


 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure- 1- Showing the digestion of carbohydrates

Absorption of monosaccharides

Only monosaccharides are absorbed  in the intestinal mucosal cells. Minute quantities of disaccharides  absorbed are rapidly eliminated through kidneys.  

The processes involved are-

1) Active transport(Sodium dependent co transport)

2) Facilitated diffusion(Carrier mediated)

3) Passive diffusion 

Glucose and galactose are absorbed by a sodium-dependent process. They are carried by the same transport protein (SGLT 1), and compete with each other for intestinal absorption. The carrier protein carries sodium along with Glucose. The flow of sodium is down the concentration gradient, while glucose is transported against the concentration gradient. The downward gradient of sodium transport drives the flow of glucose molecules. Sodium is later expelled out by the sodium pump, with utilization of energy.

This type of co transport is also utilized to reabsorb glucose from kidney tubules, involving SGLT2 Transporter. Deficiency of SGLT2 causes Renal Glycosuria  due to failure to reabsorb glucose from tubular filtrate.(Figure-2)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-2- Showing the co transport of Glucose mediated by SGLT-1/2. SGLT-1 are  present on the intestinal cells while SGLT-2 are  present on the proximal tubular cells.

Other monosaccharides are absorbed by carrier-mediated diffusion (Facilitated diffusion). Because they are not actively transported, fructose and sugar alcohols are only absorbed down their concentration gradient, and after a moderately high intake, some may remain in the intestinal lumen, acting as a substrate for bacterial fermentation.

Passive diffusion is a very slow process and is of less importance.

The absorbed glucose is transported to portal blood  from intestinal cell by specific GLUT-2 transporters (Facilitated diffusion). (Figure-3)

 


 Figure-3- Showing the absorption of monosaccharides
Q.2 -Discuss the causes, consequences and treatment of lactose intolerance

Answer- Lactose intolerance is caused by a deficiency of lactase enzyme, which is produced by the cells lining the small intestine. Disaccharides cannot be absorbed through the wall of the small intestine into the bloodstream, so in the absence of lactase, lactose present in ingested dairy products remains uncleaved and passes intact into the colon. The operons of enteric bacteria quickly switch over to lactose metabolism, and the resulting in-vivo fermentation produces copious amounts of gas (a mixture of hydrogen, carbon dioxide, and methane). This, in turn, may cause a range of abdominal symptoms, including abdominal cramps, bloating, and flatulence. In addition, as with other unabsorbed sugars (such as Sorbitol, Mannitol, and xylitol), the presence of lactose and its fermentation products raises the osmotic pressure of the colon contents.

The osmotic load of the unabsorbed lactose causes secretion of fluid and electrolytes until osmotic equilibrium is reached. Dilation of the intestine caused by the osmosis induces an acceleration of small intestinal transit, which increases the degree of maldigestion. The combined increase in fecal water, intestinal transit, and generated hydrogen gas accounts for the wide range of gastrointestinal symptoms.

Classification of Lactose Intolerance

There are three major types of lactose intolerance:

1) Primary lactose intolerance

Primary lactase deficiency develops over time and begins after about age 2 when the body begins to produce less lactase. Most children who have lactase deficiency do not experience symptoms of lactose intolerance until late adolescence or adulthood.

2) Secondary lactose intolerance

Secondary, or acquired, lactase deficiency may develop in a person with a healthy small intestine during episodes of acute illness. This occurs because of mucosal damage or from medications resulting from certain gastrointestinal diseases, including exposure to intestinal parasites such as Giardia lamblia. In such cases the production of lactase may be permanently disrupted. A very common cause of temporary lactose intolerance is gastroenteritis, particularly when the gastroenteritis is caused by rotavirus. Another form of temporary lactose intolerance is lactose overload in infants. Secondary lactase deficiency also results from injury to the small intestine that occurs with celiac disease, Crohn’s disease, or chemotherapy. This type of lactase deficiency can occur at any age but is more common in infancy.

3) Congenital lactase deficiency

It is a genetic disorder which prevents enzymatic production of lactase.  It is present at birth, and is diagnosed in early infancy.

Diagnosis

Two tests are commonly used -

Hydrogen Breath Test

The person drinks a lactose-loaded beverage and then the breath is analyzed at regular intervals to measure the amount of hydrogen. Normally, very little hydrogen is detectable in the breath, but undigested lactose produces high levels of hydrogen. The test takes about 2 to 3 hours

Stool Acidity Test

The stool acidity test is used for infants and young children to measure the amount of acid in the stool. Undigested lactose creates lactic acid and other short chain fatty acids that can be detected in a stool sample. Glucose may also be present in the stool as a result of undigested lactose.

Besides these tests, urine shows- positive test  with Benedict’s test, since lactose is a reducing sugar and a small amount of lactose is absorbed in the intestinal cell by pinocytosis and is rapidly eliminated through kidneys in to urine.

Mucosal biopsy confirms the diagnosis.

Management of lactose intolerance

Although the body’s ability to produce lactase cannot be changed, the symptoms of lactose intolerance can be managed with dietary changes. Most people with lactose intolerance can tolerate some amount of lactose in their diet. Gradually introducing small amounts of milk or milk products may help some people adapt to them with fewer symptoms. Lactose-free, lactose-reduced milk, Soy milk and other products may be recommended. Lactase enzyme drops or tablets can also be consumed. Getting enough calcium is important for people with lactose intolerance when the intake of milk and milk products is limited. A balanced diet that provides an adequate amount of nutrients—including calcium and vitamin D—and minimizes discomfort is to be planned for the patients of lactose intolerance.

 

Q.3 – Discuss the mechanism of Glucose uptake in peripheral tissues

 Answer- Glucose transporters comprise a family of at least 14 members. The most well characterized members of the family are GLUT1, GLUT2, GLUT3, GLUT4 and GLUT5.  These transporters mediate the thermodynamically downhill movement of glucose across the plasma membranes of animal cells. These are bidirectional; they can transport glucose both into and out of cells and are driven by the concentration gradient. However, export of glucose from tissues to the circulation is limited to organs that produce sugar (liver and kidney) or to organs that receive sugar from the outer milieu (the small intestine). 

The members of this family have distinctive roles:

 1. GLUT1 and GLUT3, present in nearly all mammalian cells, are responsible for basal glucose uptake. Their Km value for glucose is about 1 mM, significantly less than the normal serum-glucose level, which typically ranges from 4 mM to 8 mM. Hence, GLUT1 and GLUT3 continually transport glucose into cells at an essentially constant rate.

 2. GLUT2, present in liver and pancreatic beta cells, are distinctive in having a very high K m value for glucose (15 20 mM). Hence, glucose enters these tissues at a biologically significant rate only when there is much glucose in the blood. The pancreas can thereby sense the glucose level and accordingly adjust the rate of insulin secretion. Insulin signals the need to remove glucose from the blood for storage as glycogen or conversion into fat. The high Km value of GLUT2 also ensures that glucose rapidly enters liver cells only in times of plenty.  They are also present on serosal surface of intestinal cells and are involved in transportation of glucose from intestinal cells to portal blood.

 3. GLUT4, which has a Km value of 5 mM, transports glucose into muscle and fat cells. The presence of insulin, which signals the fed state, leads to a rapid increase in the number of GLUT4 transporters in the plasma membrane (Figure-4). Hence, insulin promotes the uptake of glucose by muscle and fat. The number of these transporters present in muscle membranes increase in response to endurance exercise training.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-4-Insulin regulates glucose uptake into these cells(They are present in skeletal, cardiac muscles and adipose tissue) by recruiting membrane vesicles containing the GLUT4 glucose transporters from the interior of cells to the cell surface, where it allows glucose to enter cells by facilitative diffusion.  Once in the cytoplasm, the glucose is phosphorylated and thereby trapped inside cells. The effect of insulin on GLUT4 distribution is reversible.  Within an hour of insulin removal, GLUT4 is removed from the membrane and restored intracellular in vesicles ready to be re-recruited to the surface by insulin.  Thus, glucose uptake by muscle and fat cells is regulated by modulating the number of GLUT4 glucose transporters on the surface of cells. 

4. GLUT5, present in the small intestine, testes, seminal vesicles and kidney, function  primarily as  fructose transporters.

5. GLUT 6- is a product of pseudo gene.

6.GLUT-7 - are  present at the surface of endoplasmic reticulum and are  related with perhaps the export of glucose from endoplasmic reticulum to cytoplasm, after the action of glucose-6 phosphatase (Old concept- recently these transporters have been found in the small intestine also)

There is increased expression of GLUT1 and GLUT3 transporters on the surface of cancer cells. Cancer cells grow more rapidly than the blood vessels to nourish them; thus, as solid tumors grow, they are unable to obtain oxygen efficiently. In other words, they begin to experience hypoxia. Under these conditions, glycolysis leading to lactic acid fermentation becomes the primary source of ATP. Glycolysis is made more efficient in hypoxic tumors by the action of a transcription factor, hypoxia-inducible transcription factor (HIF-1). In the absence of oxygen, HIF-1 increases the expression of most glycolytic enzymes and the glucose transporters GLUT1 and GLUT3. In fact, glucose uptake correlates with tumor aggressiveness and a poor prognosis.

 

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Chapter-7- Uronic acid pathway
Q.1 -Discuss the steps of Uronic acid pathway and state its biological significance?
Or
Which pathway of Glucose utilization is involved in the detoxification of Bilirubin and other foreign substances? Write the steps and elaborate on the process of detoxification of the foreign substances by the product of this pathway.
Or
Which pathway of glucose utilization is involved in the synthesis of Ascorbic acid in plants and many organisms? What is the reason that human beings and certain animals still require Ascorbic acid in their diets?
Q.2- What is the defect in essential Pentosuria? How can this be diagnosed? What are the clinical manifestations and how is this defect treated?
Or
A 35-year-old male was referred to the biochemical laboratory for the study of his mellituria. The urinary sugar had been discovered during hospitalization for the repair of an inguinal hernia when the patient was 20 years of age, and he was then refused operation because of”diabetes.” Numerous blood sugar analyses were reported normal. The patient was in excellent health and had no symptoms of diabetes, but was concerned about his condition .L- Xylulose was present excessively in urine.What is the probable diagnosis?
Chapter-8- Glycogen Metabolism
Q.1- Why do animals store energy as glycogen? Why not convert all excess fuel into fatty acids?
Q.2- What is Glycogenesis? Describe the steps and state under what conditions Glycogenesis would be promoted in the body?
Or
Describe the separate roles of Glycogenin and Glycogen Synthase in glycogen synthesis. Summarize the reactions catalyzed by each enzyme.
Q.3- Explain the pathway by which Glycogen is degraded in the body.
Or
Give a brief account of the process of Glycogenolysis. Highlight the role played by each of the participating enzymes.
Q.4- What is the role played by pyridoxal phosphate in glycogen metabolism?

Q.5- What is the cost of converting glucose 6-phosphate into glycogen and back into glucose-6-phosphate?

Or

Q.6-Is the energy required to synthesize glycogen from glucose 6-phosphate the same as the energy required to degrade glycogen to glucose 6-phosphate?

Q.7-“The phosphoroylytic cleavage of glycogen is the key for glycogen metabolism.” Justify the statement.

Q.8- Give a brief account of the regulation of glycogen metabolism.

Or

Discuss the regulation of Glycogen metabolism during well fed and fasting states.

Or

What is the effect of a high carbohydrate meal upon blood glucose and the glucose in liver cells?

Q.9 How does the regulation of phosphorylase in the liver differ from phosphorylase regulation in muscle?

Q.10-What is the effect of changes in the insulin/glucagon ratio, blood glucose or epinephrine upon glycogen synthesis and glycogen degradation in the liver?

Q.11-What is the effect of insulin upon the cAMP cascade?

Q.12-What are the three major activators of phosphorylase in muscle?

Q.13- Draw a diagram and describe the reaction cascade by which cyclic AMP alters activity of the Glycogen Phosphorylase enzyme.

Q.14- How does the rise in cytosolic [Ca++]that occurs during activation of muscle contraction affect glycogen breakdown? How is this significant with regard to metabolism during muscle contraction?

Q.15- Give a brief account of the defect,clinical presentation, laboratory diagnosis and treatment of-

a) Von-Gierke’s disease
b) McArdle’s syndrome
c) Amylopectinosis
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Chapter 6- HMP pathway

Q.1- What is HMP pathway? Explain in detail the steps of this pathway.

Q.2- Enlist the important differences between Glycolysis and HMP pathway.

Q.3- The pentose phosphate pathway is active in liver, adipose tissue,adrenal cortex, thyroid, erythrocytes, testis, and lactating mammary glands. Why is this pathway less active in nonlactating mammary glands and skeletal muscles?

Q.4- Discuss the regulation of HMP pathway.  Under what conditions is this pathway stimulated or inhibited?

Q. 5- Glucose 6-phosphate is metabolized by both the glycolytic pathway and the pentose phosphate pathway. How is the processing of this important metabolite partitioned between these two metabolic routes?

Q.6 – Discuss the metabolic significance of HMP pathway.

Q.7- What are the predominant pathways of Glucose utilization in Erythrocytes? Give the significance of each.

Q.8- Is it correct to say that excessive carbohydrate ingestion leads to obesity?

Q. 9- What is the reason that individuals with reduced ability to produce NADPH are at increased risk for specific recurrent infections?

Q.10-  What two products of the linear portion of the Pentose Phosphate Pathway(Oxidative phase) have essential roles in anabolic metabolism? What are these roles?

Or

What is the significance of oxidative phase of HMP pathway?

Q.11- If a patient has glucose-6-phosphate dehydrogenase deficiency, why are red blood cells lysed while other cells of the body remain intact? What is the biochemical basis for hemolysis? Why doesn’t this disease show up earlier in life? Give a brief account of the drug induced hemolytic anemia in G6 PD deficiency.

Or

A 34- year-old African –American man was seen with fever and shortness of breath. Shortly afterwards he developed Pancreatitis and was treated with an antibiotic, clindamycin and primaquine. After four days in to this therapy the onset of hematuria was noted. The patient’s Hb fell from 11.0g/dl to 7.4g/dl, his total Bilirubin increased from 1.2 mg/dl to 4.3 mg/dl.

What is the probable diagnosis?

What is the relationship of Primaquine and hemolytic anemia?

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Chapter-3 -Formation and Fate of Pyruvate

 Q.1- Discuss the fate of pyruvate under different metabolic conditions.

 Q.2- Give a brief account of Pyruvate dehydrogenase complex and explain the role of various factors and hormones in the regulation of this complex.

 Q.3–What could be the consequences of Pyruvate dehydrogenase complex (PDH) deficiency, explain  in detail about the causes, clinical manifestations and treatment of patients suffering from PDH complex deficiency?

 Q. 4- What is the cause of lactic acidosis in PDH complex deficiency?

Chapter 4- TCA Cycle/Citric acid Cycle/ Krebs cycle

 Q.1-What is TCA cycle?  Describe the steps and explain the reason that citric acid cycle can operate only in the presence of oxygen?

 Q.2-What would be the total energy yield when glucose is completely oxidized to CO2 and water?

 Q.3- Discuss the regulation of TCA cycle.

Or

What is respiratory control of TCA cycle?

 Q.4- What is the significance of TCA Cycle?

Or

 Discuss the amphibolic role of TCA cycle

 Q.5.- Discuss the role played by vitamins in the operation of TCA cycle

 Q6.- What are Anaplerotic reactions ?

Or

How are citric acid cycle intermediates replenished if any are drawn off for biosynthesis?

 Q.7- Why it is said that fats burn in the flame of carbohydrates?

 Q.8 -What would be the total energy yield from complete oxidation of Acetyl coA in TCA Cycle in the presence of Malonate?

 Q.9- Enlist the inhibitors of TCA cycle and mention the mechanism of action of each of them.

Chapter 5- Gluconeogenesis

 Q.1- Justify the statement -Gluconeogenesis is not a reversal of glycolysis.

 Or

 Give a brief account of the thermodynamic barriers of gluconeogenesis.

 Q.2 – How do the substrates of gluconeogenesis gain entry in to the main pathway?

 Explain giving reactions involving each substrate.

 Q.3- “It is incorrect to say that fats can not be converted to glucose”. Comment on the 

 statement.

 Q.4- What is Cori cycle or Lactic acid cycle? How does it differ from Glucose Alanine cycle? Explain how muscle may be used to supply energy in a wasting condition like anorexia nervosa.

 Q.5- Comment on the statement– ‘Glycolysis and Gluconeogenesis are reciprocally regulated’

Or

Discuss the regulation of Gluconeogenesis.

 Q.6 – What is the cause of Alcohol induced hypoglycemia?

 Q.7- Premature and low-birth-weight babies are more susceptible to hypoglycemia, what could be the possible cause for this?

 Q.8-What is the role played by kidney in gluconeogenesis?

 Q.9-What is the biochemical basis for

 a) Hypoglycemia in-Babies of diabetic mothers

 b) Maternal  hypoglycemia  during pregnancy

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Chapter-1 Digestion and Absorption of Carbohydrates

Q.1- Give a brief account of digestion and absorption of dietary carbohydrates.

Q.2- Discuss the causes, consequences, laboratory diagnosis and treatment of lactose intolerance

Q.3- Discuss the mechanism of Glucose uptake in peripheral tissues

Chapter-2 Glycolysis

Q.1- In mammals, glucose is the only fuel that the brain uses under non starvation conditions and the only fuel that red blood cells can use at all. There are many carbohydrates, Why is glucose instead of some other monosaccharides such a prominent fuel?

Q.2- Justify the statement ‘Hexokinase Traps Glucose in the Cell and begins Glycolysis’.

Q.3- What are the important differences between Hexokinase and Glucokinase?

Q.4- Give a brief account of glycolysis mentioning the steps and the energy yield per molecule of glucose.

Q.5- Discuss the regulation of Glycolysis. Why is phosphofructokinase rather than hexokinase the pacemaker of glycolysis?

Q.6 – Explain the effect of increasing the concentration of each of the following metabolites on the net rate of glycolysis: (a) glucose-6-phosphate (b) fructose-1,6-bisphosphate (c) citrate.

Q.7- Discuss the formation and degradation of Fructose 2,6 bisphosphate. What is feed forward stimulation in glycolysis?

Or

Discuss the role of Fr 2,6 bisphosphate in the regulation of glycolysis.

Or

How is the concentration of fructose 2,6-bisphosphate appropriately controlled?

Q.8- Red blood cells have an alternate pathway for glycolysis that produces an intermediate that is essential for the function of the red blood cell. This detour bypasses an ATP generating step. Discuss this detour in terms of the intermediate that is generated and the function of red blood cells.

Or

What is Luebering-Rapapport pathway? What is its significance?

Q.9 – Explain which metabolic intermediate(s) will accumulate when each of the following is added to cell-free extracts capable of glycolysis: (a) fluoride, which inhibits Enolase (b) an inhibitor of lactate dehydrogenase (c) an inhibitor of pyruvate kinase.

Q.10-Glucose-6-phosphate is at the cross roads of 3 metabolic pathways in liver cells. Name the pathways and discuss the metabolic conditions that determine which pathway will prevail.

Q.11- Justify the statement –“Muscle cells derive almost all of the ATP consumed during exertion from anaerobic glycolysis.”

Or

Why is lactate, rather than pyruvate, produced by normal muscle when it is working anaerobically?

Q.12 Are substrates other than glucose used in Glycolysis?

Q.13- What is the significance of glycolysis other than energy production?

Q.14- What is the cause of haemolytic anemia in patients suffering from Pyruvate kinase deficiency?

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Q.1-Give the details of the reaction catalyzed by each of the following enzymes-

a) Fructokinase

The conversion of Fructose to Fructose-1-phosphate is catalyzed by Fructokinase. It is the first step of fructose metabolism.

Fructose+ ATP————–>Fructose-1-phosphate+ADP

A specific kinase, Fructokinase, in liver, kidney, and intestine,catalyzes the phosphorylation of fructose to fructose 1-phosphate. This enzyme does not act on glucose, and, unlike Glucokinase, its activity is not affected by fasting or by insulin, which may explain why fructose is cleared from the blood of diabetic patients at a normal rate.

 b) Glyceraldehyde-3- phosphate dehydrogenase

The enzyme glyceraldehyde 3-phosphate dehydrogenase, is NAD+ dependent and catalyzes the oxidation of Glyceraldehyde-3-phosphate to 1, 3 bisphosphoglycerate.  

1,3-Bisphosphoglycerate is an acyl phosphate. Such compounds have a high phosphoryl-transfer potential; one of its phosphoryl groups is transferred to ADP in the next step in glycolysis. The reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase is really the sum of two processes: the oxidation of the aldehyde to a carboxylic acid by NAD+ and the joining of the carboxylic acid and orthophosphate to form the acyl-phosphate product.

Glyceraldehyde-3-phosphate+ NAD++ Pi>1, 3 bisphosphoglycerate+ NADH+ H+

The enzyme is inhibited by the —SH poison iodoacetate,which is thus able to inhibit glycolysis.  Arsenate is also inhibitor of this enzyme. The toxicity of arsenic is the result of competition of arsenate with inorganic phosphate (Pi) in the above reactions to give 1-arseno-3-phosphoglycerate,which hydrolyzes spontaneously to 3-phosphoglycerate without forming ATP.

 c) Aconitase

 Citrate is isomerized to Isocitrate by theenzyme Aconitase (aconitate hydratase); the reaction occurs in two steps: dehydration to cis-aconitate and rehydration to isocitrate.

Citrate<——->[Cis Aconitate]-———->Isocitrate

Aconitase is an iron-sulfur protein, or nonheme ironprotein. It contains four iron atoms that are not incorporated as partof a heme group. The enzyme is inhibited by Fluoroacetate. The poison Fluoroacetateis toxic, because fluoroacetyl-CoA condenses with oxaloacetate to formfluorocitrate, which inhibits Aconitase, causing citrate to accumulate. This isan example of suicidal inhibition.

 d) Malic enzyme

Malate to pyruvate conversion is catalyzedby the “malic enzyme” (NADP malate dehydrogenase). This reaction provides an alternative source of NADPH, which is used for thereductive biosynthesis.

 Malate+ NADP++Mg++——————–>Pyruvate+CO2+NADPH+H+     

 e) Xylitol dehydrogenase

 The enzyme catalyzes the conversion of L-Xylulose to Xylitol which is subsequently converted to D- Xylulose.  The enzyme is NADP dependent. The deficiencycauses Essential Pentosuria. L- Xylulose is excessively excreted inurine.

L –Xylulose+ NADPH+H+————->Xylitol————–>D-Xylulose

 f)Bisphosphoglycerate mutase

In erythrocytes, the reaction catalyzed by phosphoglyceratekinase may be bypassed to some extent by the reaction of bisphosphoglyceratemutase, which catalyzes the conversion of 1,3-bisphosphoglycerate to2,3-bisphosphoglycerate, followed by hydrolysis to 3-phosphoglycerate and Pi,catalyzed by 2,3-bisphosphoglycerate phosphatase. This alternativepathway involves no net yield of ATP from glycolysis. However, it does serve toprovide 2,3-bisphosphoglycerate, which binds to hemoglobin, decreasing itsaffinity for oxygen, and so making oxygen more readily available to tissues.

1,3- Bisphosphoglycerate———->2,3-Bisphosphoglycerate–>3-phosphoglycerate+Pi

 g) Hexokinase

 Glucose enters glycolysis by phosphorylation to glucose 6-phosphate, catalyzed by hexokinase, usingATP as the phosphate donor. Under physiologic conditions, the phosphorylation of glucose to glucose 6-phosphate can be regarded as irreversible. Hexokinaseis inhibited allosterically by its product, glucose 6-phosphate. The deficiencyof hexokinase causes hemolytic anemia.

Glucose + ATP———–>Glucose-6-phosphate

 h) UDP- Glucuronyl transferase

 Bilirubin +2UDP-Glucuronic Acid————–>Bilirubin Diglucuronide +2UDP

  The conjugation of Bilirubin is catalyzed by a specific glucuronyl transferase.The enzyme is mainly located in the endoplasmic reticulum, uses UDP-Glucuronic acid as the Glucuronyl donor, and is referred to as Bilirubin-UGT. Bilirubin Monoglucuronide is an intermediate and is subsequently converted to thediglucuronide. Most of the bilirubin excreted in the bile of mammals is in the form of bilirubin diglucuronide.

 i) UDP- G dehydrogenase- It catalyzes the conversion of UDP glucose to UDP Glucuronic acid.

In liver, the uronic acid pathwaycatalyzes the conversion of glucose to Glucuronic acid, ascorbic acid (exceptin human beings and other species for which ascorbate is a vitamin), andpentoses. Glucose 6-phosphate is isomerized to glucose 1-phosphate, which thenreacts with uridine triphosphate (UTP) to form uridine diphosphate glucose(UDPGlc) in a reaction catalyzed by UDPGlc pyro phosphorylase, as occurs in glycogen synthesis.

 Glucose—->G-6-P—–>G-1-P

 G-1-P +UTP——->UDP-Glucose+PPi

 UDPGlc is oxidized at carbon 6 byNAD-dependent. UDPGlc dehydrogenase in a two-step reaction to yield UDP-glucuronate

 UDP-Glucose+2 NAD+ ——–>UDP Glucuronic acid+ 2NADH+2H+

 UDP-glucuronate is the source ofglucuronate for reactions involving its incorporation into Proteoglycans or forreactions of substrates such as steroid hormones, Bilirubin, and a number o fdrugs that are excreted in urine or bile as glucuronide conjugates.

 j) Galactose-1-phosphate Uridyl transferase

 Galactokinase catalyses the phosphorylation of galactose, using ATP asphosphate donor

Galactose + ATP———–>Galactose-1-phosphate

 Galactose 1-phosphate reacts with uridine diphosphate glucose (UDPGlc) to form uridine diphosphate galactose (UDPGal) andglucose 1-phosphate, in a reaction catalyzed by galactose 1-phosphate uridyl transferase.

 Galactose-1-phosphate+ UDP-Glucose——————>UDP –Galactose +Glucose-1-P

The conversion of UDP Gal to UDPGlc iscatalyzed by UDPGal 4-epimerase. Inability to metabolize galactose occurs in the galactosemias, which may be caused by inherited defects of Galactokinase, uridyl transferase, or 4-epimerase, though deficiency of uridyl transferase is the best known.

Q.2- Mention the defect in each of the following diseases-

 Disease                                                      Enzyme Deficient

 a) Essential Pentosuria                                    Xylitol dehydrogenase

b) Von-Gierke’s disease                                   Glucose-6-phosphatase

c) Hereditary Fructose Intolerance           Aldolase B

d) Benign Galactosemia                                  Galactokinase

e) Classical Galactosemia                              Galactose-1-PUridyl transferase

f) Amylopectinosis                                           Branching Enzyme

g) Favism                                                             Glucose-6-phosphatedehydrogenase

h) Mc Ardle’s syndrome                                Muscle Phosphorylase

Q.3- Name the enzyme for each of the following reactions-

1) UDP Glucose—————————>UDP- Glucuronic acid.

(UDP Glucose-dehydrogenase)

 2) Phospho enolPyruvate—————> Pyruvate

(Phosphoenolpyruvate Carboxy kinase)

 3) 3-phosphoglycerate<-—————–>1,3 Bisphosphoglycerate

(Phosphoglycerate kinase)

4) Pyruvate <——————————->Lactate

(Lactate dehydrogenase)

5) Pyruvate———————————>Alanine

 (ALT- Alanine aminotransferase)

6) Pyruvate———————————> Oxalo acetate

 (Pyruvate Carboxylase)

7) Pyruvate——————————–>Acetyl co A

 (Pyruvate dehydrogenase complex)

8)Pyruvate——————————->Malic acid

 (Malic enzyme)

9)Fumarate——————————>Malate

 (Fumarase)

10)Fructose—————————–>Fructose-1-phosphate

 (Fructokinase)

11) Fructose-1-phosphate—-.>Glyceraldehyde + Dihydroxy acetone phosphate

 (Aldolase B)

12)1,3Bisphosphoglycerate————>2,3 Bisphosphoglycerate

 (BisphosphoglycerateMutase) 

13) Glucose———————————–> Sorbitol

(Aldose Reductase)

 14) Sorbitol————————————>Fructose

(Sorbitol dehydrogenase)

15) Glucose-1-P +UTP——————->UDP glucose+PPi

 (UDP-glucose pyrophosphorylase)

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Q.1-Give the details of the reaction catalyzed by each of the following enzymes

a) Fructokinase

b) Glyceraldehyde-3- phosphate dehydrogenase

c) Aconitase

d) Malic enzyme

e) Xylitol dehydrogenase

f) Phosphoglycerate mutase

g) Hexokinase

h) UDP- Glucuronyl transferase

i) UDP- G dehydrogenase

j) Galactose-1-phosphate Uridyl transferase

Q.2- Mention the defect in each of the following diseases

a) Essential Pentosuria

b) Von-Gierke’s disease

c) Hereditary Fructose Intolerance

d) Benign Galactosemia

e) Classical Galactosemia

f) Amylopectinosis

g) Favism

h) McArdle’s syndrome

Q.3- Name the enzyme for each of the following reactions

1) UDP Glucose ————- ———>  UDP-Glucuronic acid.

2) Phospho enol Pyruvate————> Pyruvate

3)  3-phosphoglycerate<————–>1,3 Bisphosphoglycerate

4) Pyruvate<—————————-> Lactate

5) Pyruvate——————————>Alanine

6) Pyruvate——————————> Oxalo acetate

 7) Pyruvate ————————–.–> Acetyl co A

8) Pyruvate——————————-> Malic acid

9) Fumarate—————————–> Malate

10) Fructose—————————–> Fructose-1- phosphate

11) Fructose-1- phosphate————> Glyceraldehyde + Dihydroxy acetone phosphate

12)1,3 Bisphosphoglycerate———->2,3 Bisphosphoglycerate    

13) Glucose——————————> Sorbitol

14) Sorbitol——————————–>Fructose

15) Glucose-1-P + UTP——————>UDP glucose+PPi

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