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Theory Notes

For Metabolism of fructose Lecture-1

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Clinical significance

1) Hereditary fructose Intolerance

Biochemical defect- Hereditary fructose intolerance is caused by mutation in the gene encoding Aldolase B enzyme.

Inheritance- It is an autosomal recessive trait that is equally distributed between the sexes.

Clinical features

These patients are healthy and asymptomatic until fructose or sucrose (table sugar) is ingested (usually from fruit, sweetened cereal, or sucrose-containing formula).

Clinical features include-

  • Recurrent vomiting,
  • Abdominal pain, and
  • Hypoglycemia that may be fatal.
  • Older patients who survive infancy develop a natural avoidance of sweets and fruits early in life and as a result frequently are without any dental caries.

Long-term exposure to fructose can result in

  • Liver failure
  • Renal tubulopathy,
  • Growth retardation

Pathophysiology- A defect in the Aldolase B gene results in a decrease in activity that is 15 percent or less than that of normal. This results in a buildup of Fructose-1-P levels in the hepatocytes.

The hypoglycemia that results following fructose uptake is caused due to

a) Inhibition of glycogenolysis, by fructose-1-phosphate, interfering with the phosphorylase action; and

b) Inhibition of gluconeogenesis at the deficient aldolase step.

Since the rate of fructose phosphorylation by fructokinase is so high, intracellular levels of both ATP and inorganic phosphate (Pi) are significantly decreased. The drop in ATP concentration adversely affects a number of cellular events, such as:

i) A hyperuricemic condition as a result of an increase in uric acid formation- See the details below (Figure 1)

ii) Severe hepatic dysfunction may be a manifestation of focal cytoplasmic degeneration and cellular fructose toxicity.

iii) Renal tubular dysfunction-The cause remains unclear; patients with renal tubular dysfunction primarily present with a proximal tubular acidosis complicated by aminoaciduria, glucosuria, and phosphaturia.

Laboratory findings

  • Urine analysis for the presence of reducing sugar- Based on the thorough dietary history of an ill child, the most straightforward approach to diagnosis of fructose 1-phosphate Aldolase deficiency is to demonstrate the presence of a non–glucose-reducing sugar in the urine. This is readily accomplished with Clinitest. Then, if test results are positive, thin-layer chromatographic separation should be used for confirmation.
  • Urine metabolic screening results may also provide evidence of glucosuria, proteinuria, and aminoaciduria, all of which are part of renal Fanconi syndrome.
  • Plasma electrolyte levels are important to determine, because the renal tubular acidosis component of hereditary fructose intolerance (HFI) may significantly depress the total plasma bicarbonate level.
  • Obtain liver function test results to assess the degree of hepatocellular disease.

Treatment

i)  Complete elimination of all sources of sucrose, fructose, and sorbitol from the diet. With this treatment, liver and kidney dysfunction improve, and symptoms become milder, even after fructose ingestion, and the long-term prognosis is good.

ii) Hepatomegaly may require months to resolve.

Complications

 Prolonged delay in diagnosis may result in cirrhotic changes with subsequent degeneration of function.

2) Essential Fructosuria

Biochemical defect- 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 the urine.

Inheritance– The inheritance is autosomal recessive.

Clinical manifestations

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 symptom less excretion of fructose in the 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.

3) Hyperuricemia upon excessive fructose ingestion

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 –1). ADP formed from the first step is converted to AMP, which is a substrate for AMP deaminase enzyme for further metabolism. 

Fructose induced hyperuricemia

Figure-1-The phosphorylation of fructose catalyzed by fructokinase is faster than subsequent cleavage by Aldolase, as a consequence ATP pool is depleted, with the resultant rapid degradation of ADP to AMP and finally to uric acid through intermediate formation of IMP, hypoxanthine and uric acid.

AMP deaminase enzyme that causes conversion of AMP to IMP (Inosine monophosphate) is regulated by inorganic phosphate. The rising concentration of inorganic phosphate inhibits this enzyme to prevent degradation of AMP. Upon excessive fructose ingestion, inorganic phosphate pool is depleted as a result the inhibition of AMP deaminase is lost. The overactive AMP deaminase converts AMP to IMP at an enhanced rate.  IMP, is subsequently converted to hypoxanthine then to xanthine and finally to uric acid. Excessive uric acid generation leads to gout or renal stones.

 AMP deaminase

Figure 2- 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 to uric acid, thus excess uric acid is formed upon excessive fructose ingestion.

4) Obesity upon excessive fructose consumption

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-3). This allows fructose to flood the pathways in the liver. High fructose consumption can lead to excess pyruvate production, causing a buildup of glycolytic intermediates and Acetyl co A.  Dihydroxyacetone phosphate (DHAP), the glycolytic intermediate, can be converted to glycerol 3-phosphate providing the glycerol backbone for the triglyceride molecule.  Excess Acetyl co A is channeled towards fatty acid synthesis.

Triglycerides , thus synthesized 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 form the basis for the development of the metabolic syndrome, hypertension, glucose intolerance and type 2 diabetes mellitus.

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.

 Fructose induced obesity

Figure-3- Hypertriglyceridemia upon excessive fructose consumption

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Digestion and absorption of fructose

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 (figure-1) transport proteins. 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 enterocytes 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 enterocytes through GLUT2 (figure-1).

Absorption of fructose

Figure-1- Absorption and transportation of fructose.

Clinical significance

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 exerts osmotic pressure, reduces the absorption of water and is metabolized by normal colonic bacteria to organic acids and the gases such as hydrogen, carbon dioxide and methane. This abnormal increase in hydrogen is detectable with the hydrogen breath test. 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 fructose containing nutrients provides symptomatic relief in a high proportion of patients.

Metabolism of fructose

Much of the ingested fructose is metabolized by the liver, using the fructose 1-phosphate pathway (Figure-2).

1) The first step is the phosphorylation of fructose to fructose 1-phosphate by fructokinase.

2) Fructose 1-phosphate is then split into glyceraldehyde and dihydroxyacetone phosphate, the intermediates of glycolysis. This aldol cleavage is catalyzed by a specific fructose 1-phosphate aldolase.

3) Glyceraldehyde is then phosphorylated to glyceraldehyde 3-phosphate, a glycolytic intermediate, by triose kinase.

Metabolism of fructose

Figure-2- Fructose metabolism by Fructose-1-P pathway

4) 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.

5) Fate of triose phosphates:

a) The two triose phosphates, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, may be oxidized by glycolysis or

b) May condense together in the presence of Aldolase to form Fructose 1, 6 bisphosphate, that may be cleaved by fructose 1, 6 bisphosphatase to  fructose-6-P.

c) Glucose-6-P  produced from Fructose-6-P by the action of Phosphohexose isomerase

i)  May be hydrolyzed to free glucose by the action of glucose-6-phosphatase, or

ii) May enter HMP pathway for the production of NADPH and pentoses, or

iii) May be converted to Glucose-1-P to be used for glycogenesis (figure-3).

Thus glucose can be produced from fructose and can contribute to blood sugar levels.

Fate of trioses

Figure-3- Interrelation of fructose and glucose metabolism. There are two isoforms of Aldolase- Aldolase A and B. Aldolase A catalyzes the cleavage of fructose 1, 6 bisphosphate in glycolytic pathway, to form two phosphorylated trioses whereas Aldolase B, concerned with fructose metabolism catalyzes the cleavage of fructose-1-P to form Glyceraldehyde and Dihydroxyacetone -P. Glyceraldehyde has to be subsequently phosphorylated for further metabolism. The fate of trioses (oxidized or used for glucose production) depends upon the cellular conditions.

To be continued….

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Regulation

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+(Figure-1) 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 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.

 Stucture of NADPH

 

Figure-1- NADPH is the reduced form of NADP+(Nicotinamide Adenine Dinucleotide Phosphate)

2) Induction and repression– The synthesis of glucose 6-phosphate dehydrogenase and 6 -phosphogluconate dehydrogenase (Figure-2) 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.

 OXIDATIVE PHASE OF HMP

Figure-2 Glucose-6-P dehydrogenase and 6- Phosphogluconate dehydrogenase  are NADP+ specific enzymes . In tissues of high NADPH use, the enzyme activities are stimulated, as the inhibition by NADPH is overcome, with the resultant overall activation of the pathway.

Significance of HMP pathway

a) Biological Significance

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 (Figure-3) 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

 Significance of NADPH

 

Figure-3- NADPH is an important biological reducing agent, required for reductive biosynthesis , macrophageal function, maintenance of RBC membrane integrity and lens transparency . 

2. To provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides, nucleic acids ATP and coenzymes (Figure-4).

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-Pformed 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 (Figure-4) .

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

 significance of HMP

Figure-4- HMP pathway is an alternative pathway of glucose utilization meant for production of NADPH,  required for reductive biosyntheses ; synthesis of ribose, required for the synthesis of nucleotides and nucleic acids and indirect source of energy through oxidation of glycolytic intermediates.

b) Clinical Significance

Glucose-6-phosphatase dehydrogenase (G6PD) deficiency

Glucose-6-phosphatase dehydrogenase (G6PD) deficiency is the most common disease-producing enzymopathy in humans

Inheritance– Inherited as an X-linked disorder

Frequency- Glucose-6-phosphatase dehydrogenase (G6PD) deficiency affects 400 million people worldwide.

Pathophysiology– 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 (Figur-6).

G-6-PD deficiency

Figure-5- NADPH produced from HMP pathway is used for reduction of oxidized glutathione that  is required for the action of glutathione peroxidase enzyme , a Selenium containing metalloenzyme. In the absence of G6PD enzyme reduced availability of NADPH causes inhibition of activity of Glutathione peroxidase , which is required for the decomposition of H2O2. Accumulated H2O2 results in triggering of free radicle chain reaction and conversion of Hb to met Hb. By both these effects, life span of red blood cells is shortened.

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.

 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.

G 6 PD deficiency

Figure-6 -Red blood cells lacking G6P dehydrogenase fail to maintain RBC integrity and undergo premature lysis.

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.

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,

Laboratory Diagnosis

The laboratory workup for glucose-6-phosphate dehydrogenase (G6PD) deficiency includes the following:

1) Measurement of enzyme activity of G6PD

2) A complete blood cell (CBC) count with the reticulocyte count to determine the level of anemia and bone marrow function.

3) Indirect bilirubinemia occurs with excessive hemoglobin degradation and can produce clinical jaundice.

4) Serum Haptoglobin levels serve as an index of hemolysis and will be decreased.

5) LDH is high and so is the unconjugated bilirubin, indicating that there is also extravascular hemolysis.

Imaging Studies

Abdominal ultrasound may be useful in assessing for splenomegaly and gallstones in cases of glucose-6-phosphate dehydrogenase (G6PD) deficiency.

Peripheral Blood Film

The blood film shows anisocytosis, polychromasia, and spherocytes. The most typical feature is the presence of bizarre poikilocytes with red cells that appear to have unevenly distributed hemoglobin and red cells that appear to have had parts of them bitten away (bite cells or blister cells (Figure-7).

g6 pd -PBF

Figure-7- Acute hemolysis from glucose-6-phosphate dehydrogenase deficiency is linked to the development of Heinz bodies, which are composed of denatured hemoglobin

Treatment

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.When acute HA develops and once its cause is recognized, no specific treatment is needed in most cases. However, if the anemia is severe, it may be a medical emergency, especially in children, requiring immediate action, including blood transfusion.

Diet

Patients must avoid broad beans (ie, fava beans). Favism occurs only in the Mediterranean variety of glucose-6-phosphate dehydrogenase (G6PD) deficiency.

Prognosis

Most individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency do not need treatment.

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For HMP Pathway -Lecture-1

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Non Oxidative phase (Reversible phase of HMP pathway)

The pentose phosphate pathway (hexose monophosphate shunt) is a more complex pathway than glycolysis.It is presumed that three molecules of glucose-6-P enter simultaneously to give rise to three molecules of CO2 and three five-carbon sugars. These are rearranged to regenerate two molecules of glucose 6-phosphate and one molecule of the glycolytic intermediate, glyceraldehyde– 3 -phosphate (Figure–1).

Reactions of HMP pathway

Figure-1- 3 molecules of Glucose-6-P enter simultaneously in this pathway to produce 3 molecules of CO2, 6 NADPH, 2 fructose-6-P and one molecule of glyceraldehyde-3-P. 2 molecules of Fructose-6-P are converted to 2 molecules of Glucose-6-P while  Glyceraldehyde-3-P is presumed to be equivalent to half a molecule of Glucose-6-P. Three carbons less are presumed to be lost as CO2. If 6 molecules enter at the same time, then it would represent loss of 6 molecules of CO2 equivalent to complete oxidation of one molecule of glucose.

Details of reactions

Step-1- The reaction is catalyzed by Transketolase (Figure-1 and 2)

The net reaction is-

 Reaction of Transketolase

Transketolase catalyzes the transfer of the two-carbon unit comprising carbons 1 and 2 of a ketose (from Xylulose 5-phosphate) to the aldehyde carbon of an aldose sugar (Ribose 5-phosphate), producing the seven-carbon ketose sedoheptulose 7-phosphate and the aldose glyceraldehyde 3-phosphate. 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 (Figure-2). The reaction requires Mg2+ and thiamine pyrophosphate (vitamin B1) as coenzyme. The two-carbon moiety transferred is probably glycoaldehyde bound to thiamine pyrophosphate.

 Transketolase-1

Figure-2- Two phosphorylated pentoses (Keto and aldo pentoses) rearrange by transfer of two carbon units from Keto pentose (Xylulose-5-P) to Aldopentose (Ribose-5-P) to form phosphorylated aldo triose (Glycerladehyde-3-P) and phosphorylated keto heptose (Sedoheptulose-7-P). The reaction is reversible and is catalyzed by TPPTransketolase dependent enzyme.

Clinical significance- R.B.C Transketolase activity is measured to diagnose underlying thiamine deficiency, since the enzyme is TPP dependent. In thiamine deficiency Transketolase activity is reduced.

Step- 2- The reaction is catalyzed by Transaldolase enzyme (Figure-1 and 3)

The net reaction is represented as follows-

 Reaction of Transaldolase

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-3)

 Transaldolase

Figure 3-  Transaldolase catalyzed reaction involves the rearrangement of Phosphorylated trio aldose (Glyceraldehyde-3-P) and keto heptose (Sedoheptulose-7-P) by shifting of 3 carbon units to form keto hexose (Fructose-6-P) and aldo tetrose (Erythrose-4-P).

Step-3- The reaction is catalyzed by Transketolase enzyme (Figure-1 and 4)

 

Transketolase-2

In this reaction catalyzed by transketolase, Xylulose 5-phosphate again 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 4)

 

 Transketolase

Figure-4- Transketolase catalyzes the interconversion of  phosphorylated aldotetrose (C4) and ketopentose( C5) to form glycolytic intermediates , fructose-6-P (C6 ) and glyceraldehyde-3-P (C3).

Since the reactions of non oxidative phase are irreversible, the glycolytic intermediates can also rearrange to form pentoses.

The sum of these reactions is

 Net reaction of non oxidative phase

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-

 Conversion of ribose-5-P to glycolytic intermediates

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.

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The pentose phosphate pathway (also called Phospho gluconate pathway or hexose monophosphate shunt [HMP shunt]) is an alternative route for the metabolism of glucose.

Characteristics of HMP pathway

1) All the reactions of this pathway take place in the cytosol.

2)  It does not lead to formation of ATP but has two major functions: (a) The formation of NADPH for synthesis of fatty acids and steroids, and (b) the synthesis of ribose for nucleotide and nucleic acid formation.

3) HMP pathway is highly active in rapidly dividing cells and in tissues where there is a great requirement of NADPH.

4) 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.

5) Erythrocytes utilize the reactions of the PPP to generate large amounts of NADPH used in the maintenance of membrane integrity 

6) In skeletal, muscle, this pathway is less active.

7) All the intermediates of this pathway are in the mono phosphate form contrary to glycolysis where bisphosphate forms of intermediates are also there.

Overview of HMP pathway

This pathway consists of two phases: the oxidative (irreversible) phase and the nonoxidative (reversible) phase.

1) In the oxidative phase, there is oxidative decarboxylation of Glucose-6-P to form NADPH and Ribulose 5-phosphate. Ribulose is further isomerized to form Ribose-5-P. This five-carbon sugar and its derivatives are components of RNA and DNA, as well as ATP, NADH, FAD, and coenzyme A.

 

 HMP pathway -Phase-1

2) 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 degradation of excess five-carbon sugars into intermediates of the glycolytic pathway.

 Phase 2 of HMP pathway

 Over view of HMP pathway

 Over view of HMP pathway

Figure-1- HMP pathway consists of two phases, oxidative phase that leads to formation of Ribose-5-P by oxidative decarboxylation and Non oxidative phase that involves rearrangement process with the resultant formation of glycolytic intermediates. Glyceraldehyde-3-P(GAP) and Fr-6-P are intermediates of glycolysis. In other words, the glycolytic intermediates can also rearrange to form Pentoses due to reversible nature of this phase and this holds true in skeletal muscle.

 Difference between Glycolysis and HMP pathway

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

 Reactions of HMP pathway

 1) Oxidative Phase

  • The oxidative phase of the pentose phosphate pathway starts with the dehydrogenation of glucose 6-phosphate at carbon 1,  in a reaction catalyzed by glucose 6-phosphate dehydrogenase (Figure 2).
  • Unlike glycolysis, oxidation is achieved by dehydrogenation using NADP+, not NAD+, as the hydrogen acceptor.
  • 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 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 (Figure-2)

 Reactions of oxidative phase

 Figure 2- Reactions of oxidative phase of HMP pathway. 2 molecules of NADPH and one of CO2 are produced in the oxidative phase. Glucose-6-P dehydrogenase is the key regulatory enzyme of this 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)

 Fate of Ribulose-5-P           

Figure 3- Interconversion of pentoses. Ribulose-5-P is converted to by phosphopentose isomerase to form Ribose-5-P. It is aldose ketose isomerization. It is also converted to Xylulose-5-P by epimerization. The reaction is catalyzed by Phosphopentose epimerase.

To be continued in the next post…….

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Introduction

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

The entry of glucose is in the phosphorylated form (Glucose-6-P). The phosphorylation is catalyzed by Hexokinase/ Glucokinase.

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 and 5)

 Glucuronic acid synthesis

Figure-1- UDP Glucuronic acid (active form) produced from Glucose-6-P is required for the synthesis of mucopolysaccharides, proteoglycans and is also used for the detoxification of foreign compounds.

 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 and 5)

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  (Figure-2 and 5)

Synthesis of Ascorbic acid

This pathway is used by plants and some animals for the synthesis of Ascorbic acid.

L- Gulonate is dehydrated in the presence of enzyme, Aldonolactonase to form L-gulono-δ-location, 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 the enzyme L-gluconolactone oxidase leads to the formation of 2-keto gulono lactone and that is finally converted to L ascorbic acid. (Figure-2)

 Synthesis of ascorbic acid

Figure 2- 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.

In humans and other primates, as well as 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 a 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 the inefficiency to synthesize ascorbic acid in primates.

4) Fate of L- Gulonate in human beings

The Uronic acid pathway is connected to Pentose phosphate pathway through L-gulonate, (Figure-3 and 5) since the latter can be converted to an intermediate of the Pentose phosphate pathway as follows-

 Fate of L gulonate in human beings

Figure-3- L- Gulonate is oxidatively decarboxylated to form L-xylulose in human beings

5) Fate of L-xylulose

L-Xylulose is converted to the D isomer by an NADPH-dependent reduction to Xylitol, catalyzed by Xylitol dehydrogenase enzymes. The deficiency of Xylitol dehydrogenase causes Essential pentosuria, a clinical state of excess excretion of L- Xylulose in urine.

Xylitol is converted in an NAD+dependent reaction to  form D-Xylulose. The reaction is catalyzed by Xylulose reductase enzyme. D- Xylulose is then phosphorylated to D-Xylulose 5-phosphate and that is metabolized via the pentose phosphate pathway (Figure-4 and 5).

 Fate of L-Xylulose

Figure- 4- Fate of L- Xylulose. The flow of electrons is from NADPH to NAD+. Xylulose is phosphorylated by Xylulose kinase to form Xylulose-5- phosphate.

Summary of uronic acid pathway

 Uronic acid pathway

Figure-5- Uronic acid pathway is a source of glucuronides required for detoxification of drugs as well as hormones and bilirubin. It is also a source of ascorbic acid (not in human beings). Both xylulose as well as ascorbic acid can be metabolized to form oxalates.

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.

UDP-Glucuronic acid is the Glucuronyl donor, and a variety of glucuronosyl transferases, present in both the endoplasmic reticulum and cytosol, are the catalysts. Molecules such as 2-acetylaminofluorene (a carcinogen), aniline, benzoic acid, meprobamate (a tranquilizer), phenol, and many steroids are excreted as glucuronides. The glucuronide may be attached to oxygen, nitrogen, or sulfur groups of the substrates. Glucuronidation is probably the most frequent conjugation reaction.

Conjugation of Bilirubin-Bilirubin is nonpolar and would persist in cells (e.g., bound to lipids) if not rendered water-soluble. Hepatocytes convert bilirubin to a polar form, which is readily excreted in the bile, by adding Glucuronic acid molecules to it. This process is called conjugation.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 glucuronosyl donor, and is referred to as bilirubin-UGT. Bilirubin Monoglucuronide is an intermediate and is subsequently converted to the diglucuronide. Most of the bilirubin excreted in the bile of mammals is in the form of bilirubin diglucuronide (Figure-6).

 Conjugation of bilirubin

Figure-6-  Conjugation of bilirubin takes place in the liver. The reaction is catalyzed by UDP glucuronyl transferase. Bilirubin diglucuronide is the water-soluble, conjugated form of bilirubin.

 2) Synthesis of Mucopolysaccharides- Glucuronic acid is also required for the synthesis of Mucopolysaccharides such as hyaluronic acid and heparin, which contain glucuronic acid as an essential component.

Clinical Significance of Uronic acid pathway

A) Diminished activity of Bilirubin UDP Glucuronyl Transferase (UGT)

1) Neonatal “Physiologic Jaundice

This transient condition is the most common cause of unconjugated hyperbilirubinemia. It results from an accelerated hemolysis around the time of birth and an immature hepatic system for the uptake, conjugation, and secretion of bilirubin. Not only is the bilirubin-UGT activity reduced, but there probably is reduced synthesis of the substrate for that enzyme, UDP-glucuronic acid. Since the increased amount of bilirubin is unconjugated, it is capable of penetrating the blood-brain barrier when its concentration in plasma exceeds that which can be tightly bound by albumin (20–25 mg/dL). This can result in a hyperbilirubinemic toxic encephalopathy, or kernicterus, which can cause mental retardation. Because of the recognized inducibility of this bilirubin UGT enzyme system, phenobarbital has been administered to jaundiced neonates and is effective in this disorder. In addition, exposure to blue light (phototherapy) promotes the hepatic excretion of unconjugated bilirubin by converting some of the bilirubin to other derivatives such as maleimide fragments and geometric isomers that are excreted in the bile.

2) Crigler-Najjar Syndrome, Type I; Congenital Nonhemolytic Jaundice

Type I Crigler-Najjar syndrome is a rare autosomal recessive disorder. It is characterized by severe congenital jaundice (serum bilirubin usually exceeds 20 mg/dL) due to mutations in the gene encoding bilirubin-UGT activity in hepatic tissues. The disease is often fatal within the first 15 months of life. Children with this condition have been treated with phototherapy, resulting in some reduction in plasma bilirubin levels.

3) Crigler-Najjar Syndrome, Type II

This rare inherited disorder, also results from mutations in the gene encoding bilirubin-UGT, but some activity of the enzyme is retained and the condition has a more benign course than type I. Serum bilirubin concentrations usually do not exceed 20 mg/dL. Patients with this condition can respond to treatment with large doses of phenobarbital.

4) Gilbert Syndrome

Again, this relatively prevalent condition is caused by mutations in the gene encoding bilirubin-UGT. It is more common among males. Approximately 30% of the enzyme’s activity is preserved and the condition is entirely harmless.

B) Essential Pentosuria

Essential 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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Three mechanisms are responsible for regulating the activity of  key enzymes of gluconeogenesis –

(1) Changes in the rate of enzyme synthesis (Induction/Repression)

(2) Covalent modification by reversible phosphorylation, and

(3) Allosteric effects. 

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.

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 hyperglycemia after meals, stimulates the expression of glycolytic enzymes such as phosphofructokinase-1, pyruvate kinase, and the bifunctional enzyme (PFK-2 – Fr-2,6 bisphosphatase) that makes and degrades F-2,6-BP. Thus insulin stimulates glycolysis and inhibits gluconeogenesis.

Glucagon, which rises during starvation, inhibits the expression of glycolytic enzymes and stimulates instead the production of two key gluconeogenic enzymes, phosphoenolpyruvate carboxy kinase and fructose 1,6-bisphosphatase. As a result glycolysis is inhibited and gluconeogenesis is stimulated.

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 mainly two enzymes –

1) Pyruvate kinase- Pyruvate kinase exists in two forms, phosphorylated (inactive ) and dephosphorylated (active) forms. Insulin activates this enzyme by causing dephosphorylation  through stimulating phosphatase enzyme, while glucagon inactivates this enzyme by bringing about c AMP mediated phosphorylation (Figure-1).

In the inhibited state of pyruvate kinase, phosphoenol pyruvate is channeled towards glucose production .

 Reaction catalyzed by pyruvate kinase

Figure- 1-Pyruvate kinase catalyzes the conversion of phosphoenol pyruvate to pyruvate. This is a regulatory enzyme and catalyzes the last step of glycolysis.ATP is produced at this step by substrate level phosphorylation. The enzyme is inhibited by excess of ATP and alanine while it is stimulated by Fr, 1,6 bisphosphate the product of PFK-1 catalyzed reaction.

2) Bifunctional enzyme (PFK-2- Fr 2,6 bisphosphatase)– The second enzyme to be affected by phosphorylation cascade is phosphofructokinase-2 that is inactivated but Fr 2,6 bisphosphatase becomes active upon phosphorylation (Figure-2 ). As a result net concentration of fructose 2,6 bisphosphate is lowered , Phosphofructo kinase-1 enzyme gets inhibited but Fr 1,6 bisphosphatase enzyme gets stimulated (See the details below) .

 

 Reciprocal regulation of glycolysis and gluconeogenesis

Figure-2-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. 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.  

3) Allosteric Modification

It is an instantaneous process.

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. This means that as acetyl-CoA is formed from pyruvate, it automatically ensures the provision of oxaloacetate and, therefore, its further oxidation in the citric acid cycle, by activating pyruvate carboxylase (Figure-3). 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-3). 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 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-2). 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-2).

 

 Allosteric regulation

Figure-3- Reciprocal Regulation of Gluconeogenesis and Glycolysis in the Liver. PFK-1 and Fr1.6 bisphophase are reciprocally regulated. The negative modifiers of PFK-1(ATP and citrate act as positive modifiers of Fr1,6 bisphosphatase enzyme. Similarly is the case of positive modifiers of PFK-1 which act as negative modifiers of Fr1,6 bisphosphatase. Another regulation is at the level of Pyruvate kinase and pyruvate carboxylase enzymes. Pyruvate carboxylase is stimulated by Acetyl co A and inhibited by ADP while pyruvate kinase(glycolytic enzyme)is inhibited by ATP and Alanine but stimulated by ADP (low energy state).

Summary

Gluconeogenesis is stimulated under conditions of fasting and starvation under the effect of glucagon and catecholamines. The regulation is mainly brought about by induction/repression , covalent and allosteric modifications. The key enzymes of regulation are pyruvate carboxylase , phospho enol pyruvate carboxy kinase and fr1,6 bisphosphatase which are stimulated if there is need for glucose production . These effects are reversed in the fed state in the presence of insulin where the glycolytic enzymes are stimulated to promote glucose utilization and enzymes of gluconeogenesis are inhibited. Thus glycolysis and gluconeogenesis do not occur at the same pace at the same time, they are reciprocally regulated.

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

(For lactate, glycerol and propionatecheck lecture-1)

http://www.namrata.co/substrates-of-gluconeogenesis-lecture-1/

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. Acetyl co A cannot be termed glucogenic, since the conversion back to pyruvate is not possible due to irreversible nature of the reaction and in TCA cycle Acetyl co A loses both of its carbons as carbon dioxide, hence there is nothing left to contribute to glucose production.

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-1). The transamination of alanine directly yields pyruvate.

 Reaction catalyzed by ALT

Biological Significance

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-1)

 Glucose Alanine cycle

 

Figure-1- Glucose alanine cycle serves two purposes, firstly ammonia disposal (as  amino group of alanine and secondly transport of pyruvate(carbon skeleton of alanine) for glucose production.

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

Gluconeogenesis requires a coordinated supply of precursors from muscle and adipose tissue to the liver (and kidneys).

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.

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).

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.

2) Oxalo acetate- Aspartate and asparagine are converted into oxaloacetate, a citric acid cycle intermediate (Figure-2). Aspartate, a four-carbon amino acid, is directly transaminated to oxaloacetate.The reaction is catalyzed by AST (Aspartate transferase also called SGOT- Serum glutamate oxaloacetate transaminase)

 Reaction catalyzed by AST

3) α-Ketoglutarate is the point of entry of several five-carbon amino acids that are first converted into glutamate (Figure-2) . Arginine, histidine , proline, glutamate and glutamine enter the pathway as α-Ketoglutarate which is eventually converted to oxaloacetate to proceed further in to the main pathway of glucose production.

All the intermediates of TCA cycle beyond α-Ketoglutarate are considered glucogenic.

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.

The TCA intermediates either directly form pyruvate from Malate by the activity of Malic enzyme or are converted to oxaloacetate by the activity of Malate dehydrogenase so as to be converted to phosphoenol pyruvate by the activity of phospho enol pyruvate carboxy kinase enzyme.

 GLUCOGENIC AMINO ACIDS

Figure- 2- 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.

Role of kidney in gluconeogenesis

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.

During fasting (starvation) or acidosis, cortisol acts to induce muscle protein degradation. Muscle glutamine synthetase activity is induced. 

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.  Alanine and glutamine account for approximately 50% of all amino acids that leave muscle. Through the actions of transaminases, a mole of waste ammonia is transferred to α-ketoglutarate via the glutamate dehydrogenase catalyzed reaction yielding glutamate (Figure-3).

 Reaction catalyzed by glutamate dehydrogenase

Figure-3- The reaction catalyzed by glutamate dehydrogenase is reversible. The enzyme is unique in the sense that it can utilize either  of NAD + or NADP+

Glutamate is then a substrate for glutamine synthetase, which incorporates another mole of waste ammonia generating glutamine (Figure-4) .

 Reaction catalyzed by glutamine synthetase

Figure-4-  Muscle Glutamine synthetase activity increase during conditions of starvation and stress.  The activity of Brain isoenzyme increase in conditions of ammonia intoxication in order to detoxify ammonia.

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 (Figure-5).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.

 

 role of kidney in gluconeogenesis

Figure-5- Reaction 1 and 2 show the reversible reactions catalyzed by glutamate dehydrogenase enzyme. Reaction 3 for the formation of glutamine is catalyzed by Glutamine synthetase while the reaction 4 for the formation of glutamate from glutamine is catalyzed by glutaminase enzyme. Glutamine in kidney is converted first to glutamate and then to alpha ketoglutarate so as to be converted to oxaloacetate and that is converted to glucose by a series of steps.

The secretion of cortisol hormone, increases in response to a variety of stresses, including fasting and acidosis.

Revision of steps of gluconeogenesis – figure-6

 

Barriers of gluconeogenesis

 Figure-6- steps of gluconeogenesis

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The major substrates of gluconeogenesis are-

1) Lactate

2) Glycerol

3) Propionate

4) Glucogenic amino acids

The entry of non carbohydrate precursors into the pathway of glucose production is as follows-

1) Lactate– Lactate is a major source of carbon atoms for glucose synthesis by gluconeogenesis. It is formed by active skeletal muscle when the rate of glycolysis exceeds the rate of oxidative metabolism. It is also the end product of glycolysis in red blood cells and in the cells deprived of mitochondria. Lactate is readily converted into pyruvate by the action of lactate dehydrogenase (Figure-1).

 Lactate to pyruvate conversion

Figure-1- Reversible Lactate to pyruvate conversion depends upon the availability of NAD+ or NADH

Biological significance

This reaction serves two critical functions during anaerobic glycolysis.

1) 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.

 Lactic acid fermentation

Figure-2- The coupling of reactions catalyzed by Glyceraldehyde-3-Phosphate dehydrogenase and Lactate dehydrogenase allows the glycolysis to continue making the constant availability of NAD

2) Cori’s cycle- The lactate produced by the LDH reaction is released into 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 (Figure-3).

Corri cycle

Figure-3- 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.

2) Glycerol-The hydrolysis of triacylglycerols in fat cells yield 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 (see details below).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) (Figure-4).

 

 Glycerol activation

Figure-4- Activation of glycerol is needed for subsequent esterification to form triglycerides.

Glycerol kinase is absent in adipose tissue, glycerol released by hydrolysis of triglycerides  cannot be utilized for reesterificaton,  hence 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.

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).

3) Propionate– Propionate is a major precursor of glucose in ruminants; it enters gluconeogenesis via the citric acid cycle. 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 (Figure-5)

 Fate of propionate

Figure- 5- After esterification with CoA, Propionyl-CoA is carboxylated to D-Methylmalonyl-CoA, catalyzed by Propionyl-CoA carboxylase, a biotin-dependent enzyme. 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.

Methylmalonyl CoA Isomerase/ mutase is a vitamin B12 dependent enzyme, and in deficiency methylmalonic acid is excreted in the urine (methylmalonic aciduria).

Role of fatty acids in glucose production-

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 the 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 cannot 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-6),  Thus, explaining why even chain fatty acids do not undergo a net conversion of carbohydrate.

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

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

 

 Conversion of glucose to fats

Figure- 6- Glucose can be converted to Fatty acids, but due to irreversible reaction of conversion of pyruvate to acetyl Co A and the loss of both the carbons of acetyl Co A in TCA cycle as CO2, even chain fatty acids that yield Acetyl Co A upon oxidation cannot be considered glucogenic.

Summary (Figure-7)

1) Lactate enters as pyruvate

2) Glycerol enters as Dihydroxy acetone phosphate

3) Propionate enters as Succinyl Co A

4) Glucogenic amino acids (To be covered in the next post)

 

 Barriers of gluconeogenesis

 

Figure -7- steps of gluconeogenesis and entry of various non carbohydrate precursors into the main pathway for glucose production

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Gluconeogenesis is the process of converting non carbohydrate precursors to glucose or glycogen.

Substrates of Gluconeogenesis

The major substrates are-

a) The glucogenic amino acids,

b) Lactate

c) Glycerol, and

d) Propionate.

These noncarbohydrate precursors of glucose are first converted into pyruvate or enter the pathway at later intermediates such as oxaloacetate and Dihydroxyacetone phosphate.

Sites

Liver and kidney are the major gluconeogenic tissues.

Significance

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 (Figure-1)

 Barriers of gluconeogenesis

Figure-1- Barriers of gluconeogenesis. Three irreversible reactions of glycolysis are substituted by alternative reactions. Pyruvate carboxylase, Phospho enol pyruvate carboxy kinase, Fructose 1,6 bisphosphatase and glucose-6-Phosphatase enzymes are unique to the pathway of gluconeogenesis. Lactate enters as pyruvate, glycerol as Dihydroxy acetone-phosphate and propionate as Succinyl co A.  Acetyl Co A  is not glucogenic but it is a positive modulator of pyruvate carboxylase enzyme.

Details of reactions

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 carboxy kinase.

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.

 Reaction catalyzed by Pyruvate carboxylase

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 (Figure-2). 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.

 First barrier of gluconeogenesis

Figure-2- 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.

 Conversion of oxaloacetate to phosphoenol pyruvate

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.  

 

 Second barrier of gluconeogenesis

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 (Figure-3). 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.

 Third barrier of gluconeogenesis

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.

 

 Conversion of glucose-6-phosphate to glucose

Figure- 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 (Figure-3) .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. 

 

To be continued in the next post

 

 

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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 gluconeogenesis, fatty acid , haem and nucleotide biosynthesis. 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 (Figure-1).

 Catabolic role of TCA cycle

Figure-1 – All the major nutrients are completely oxidized in TCA cycle to yield energy.The coenzymes thus reduced in the process of oxidation of substrates are regenerated in the oxidized form through the involvement of electron transport chain.

Portal of entry of nutrients- The nutrients can enter  either at the level of Acetyl co A or at the level of any of intermediates of TCA cycle.  Broadly speaking carbohydrates enter as acetyl Co A through first formation of pyruvate; fatty acids enter eother as acetyl co A (even chain) or Succinyl co A (odd chain fatty acids), whereas amino acids enter at various points in TCA cycle (Figure-2) for complete oxidation or for the synthesis of biological compounds as per need of the cell. The details are as follows-

1) Pyruvate- The major sources of pyruvate are

a) Glucose- is a representative of carbohydrates,  it is oxidized through glycolysis to produce pyruvate.

Glucose is the major source of pyruvate (Figure-1 and 2).

b) Amino acids- Pyruvate forming amino acids are Glycine, serine, hydroxy proline, threonine, alanine, tryptophan and alanine (Figure-2)

c) Lactate- Pyruvate is formed from lactate under aerobic conditions . The reaction is catalyzed by lactate dehydrogenase.

Pyruvate is subsequently converted to Acetyl co A. The reaction is catalyzed by pyruvate dehydrogenase complex.

2) Acetyl co A- The major sources of Acetyl co A are as follows-

a) Fatty acids- The even chain fatty acids upon oxidation produce acetyl co A.

b) Ketone bodies – Ketone bodies upon breakdown produce acetyl co A.  Thus acetyl co A is both a precursor as well as the end product of metabolism of ketone bodies.

c) Amino acids- Leucine, Lysine, Isoleucine , Tryptophan, Tyrosine and Phenyl alanine produce acetyl co A directly or indirectly (Figure-2).

 Catabolism of amino acids

Figure-2-  The carbon skeleton of various amino acids can gain entry at different points in the TCA cycle for complete oxidation

d)  Acetyl CholineUpon hydrolysis of acetyl choline acetyl co A is produced.

e) Alcohol- Alcohol is metabolized first to acetaldehyde and then to acetate that is subsequently converted to acetyl co A.

3) α- Keto glutarate- Various amino acids like glutamate, arginine, histidine ,proline and glutamine end up their metabolism forming alpha keto glutarate.

4) Succinyl co A

a) Amino acids–  Valine, Isoleucine and methionine form Succinyl co A upon catabolism of their carbon skeleton. Valine directly forms Succinyl co A while methionine and Isoleucine first form propionyl co A that is subsequently converted to Succinyl co A (Figure-3).

b) Odd chain fatty acids- Propionyl co A produced from the metabolism of odd chain fatty acids is subsequently converted to Succinyl co A.

c) Side chain of cholesterol- During bile acid synthesis Propionyl co A is produced from the side chain of cholesterol that  gains entry in TCA cycle as Succinyl co A.

 Fate of propionyl co A

Figure-3- Fate of propionyl co A. Propionyl co A is first carboxylated to form D- Methyl malonyl co A, that is is isomerized to form L methyl malnyl coA which is finally converted to Succinyl co A.

5) Fumarate-  Phenyl alanine and tyrosine are Fumarate forming amino acids (Figure-2).

6) Oxaloacetate- Aspartate and Asparagine form Oxaloacetate that can further be utilized for oxidation or glucose production as per need of the body (Figure-2).

Anabolic  role/Significance of TCA cycle intermediates

1) Acetyl co A– It has a central role to play both in the catabolism as well as synthesis of various biological compounds. Acetyl co A is a precursor for the synthesis of-

a) Fatty acids

b) Cholesterol

c) Ketone bodies

d) Steroids

e) Acetyl choline

f) Also used for detoxification of xenobiotics

2) Citrate- Besides acting as a negative allosteric modifier for Phosphofructokinase-1 to inhibit glycolysis, it also regulates pathway of fatty acid biosynthesis by- i) stimulating the activity of  Acetyl co A carboxylase acting as a positive modifier (Citrate converts the enzyme from an inactive dimer to an active polymeric form ) and also -ii) by transporting acetyl co A to the cytoplasm from mitochondrion since the fatty acid synthesis is a cytoplasmic process (Figure-4).

 Role of citrate

Figure-4- Acetyl-CoA is formed from glucose via the oxidation of pyruvate within the mitochondria. However, it does not diffuse readily into the extra mitochondrial cytosol, the principal site of fatty acid synthesis. Citrate, formed after condensation of acetyl-CoA with Oxaloacetate in the citric acid cycle within mitochondria, is translocated into the extra mitochondrial compartment via the tricarboxylate transporter, where in the presence of CoA and ATP it undergoes cleavage to acetyl-CoA and Oxaloacetate catalyzed by ATP-citrate lyase. The acetyl-CoA is then available for malonyl-CoA formation and synthesis to palmitate.

3) Isocitrate–  Dehydrogenation of Isocitrate forms the first link between TCA cycle and electron transport chain. Cytosolic Isocitrate dehydrogenase enzyme activity provides NADPH for the reductive biosynthesis.

4) α- Ketoglutarate – Alpha ketoglutarate forms the  first link between TCA cycle and amino acid metabolism (Figure-5).

Upon transamination, glutamate is produced from alpha keto glutarate. Glutamate can be-

a) Decarboxylated to form GABA (gamma amino butyric acid) which is a neurotransmitter

b) Aminated to form Glutamine which is used for the synthesis of pyrimidine nucleotides.

 Amphibolic role of TCA cycle

Figure-5- Involvement of the citric acid cycle in fatty acid, sterol, haem, purine and pyrimidine  biosynthesis, transamination, and gluconeogenesis.

5) Succinyl co A- It is used for-

a) Utilization of ketone bodies

b) Haem biosynthesis

6) Fumarate- Forms a link between urea cycle and TCA cycle.

7) Malate is used for the transportation of-

a) Oxaloacetate from mitochondrion to cytoplasm for channeling it in to the pathway of gluconeogenesis.

b) For transportation of reducing equivalents from cytoplasm to mitochondrion (Malate Aspartate shuttle).

8) Oxaloacetate- is used not only as a biocatalyst for stimulation of TCA cycle activity but it is also used for-

a) Glucose production in the pathway of gluconeogenesis

b) Formation of Aspartate by transamination

c) Aspartate is further used for the synthesis of purine and pyrimidine nucleotides

d) Aspartate can also be used for the synthesis of Asparagine.

e) Aspartate is also used for urea formation in urea cycle.

Summary of significance of TCA cycle

Role TCA intermediate Significance
a) Catabolic Role Acetyl co A Oxidation of  carbohydrate, fatty acids, amino acids, ketone bodies and alcohol
  TCA cycle intermediates such as Alpha keto glutarate, succinyl co A, Fumarate and oxaloacetate Oxidation of carbon skeleton of various amino acids
Each Acetyl co A yields 12 ATP molecules
b) Anabolic Role Acetyl co A Synthesis of fatty acids, cholesterol, ketone bodies, steroids, acetyl choline and used for detoxification
  Citrate Promotes activity of acetyl co A carboxylase and transports acetyl co A outside the mitochondrial for extra mitochondrial fatty acid synthesis.
  α- Keto glutarate Synthesis of Glutamate, GABA and Glutamine. GABA is a neurotransmitter whereas glutamine is required for pyrimidine biosynthesis.
  Succinyl co A Utilization of ketone bodies and haem biosynthesis
  Fumarate Link between urea and TCA cycles
  Malate Transporter of Oxaloacetate and reducing equivalents
  Oxaloacetate Substrate of gluconeogenesis, forms Aspartate and Asparagine.Aspartate is needed for purine, pyrimidine and urea synthesis

Clinical Significance

Thus TCA cycle is vital to life due to its amphibolic role, no TCA cycle enzyme deficiency has yet been reported , perhaps this is not compatible with life.

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 Regulation of the TCA cycle occurs at the level of-

A) Entry of substrates into 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.

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 (Figure-1) . 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.

 Regulation of PDH complex

Figure -1-Regulation of PDH complex by covalent modification. Activation of PDH kinase causes inactivation of PDH, whereas activation of  PDH phosphatase bring about activation of enzyme

B) Key reactions of the cycle– Key regulation of the cycle occurs by regulation of the individual enzymes of TCA cycle and by respiratory control-

i) Regulation of TCA cycle enzymes-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 (Figure-2) .

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 when the cell has a high level of ATP (Figure-2).

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

 Regulation of TCA cycle

 

Figure-2- 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.

ii) Respiratory control of TCA cycle- 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.

 a) Low energy state- Under conditions of low energy state, TCA cycle activity is stimulated to restore the energy balance. This mechanism of regulation can be explained as follows-

  • Low  ATP/High ADP concentration
  • More ADP available for phosphorylation
  • Higher rate of oxidative phosphorylation
  • Higher rate of electron transport
  • More availability of oxidized coenzymes
  • More active TCA cycle enzymes

 b) High energy state- Under conditions of high energy state, TCA cycle activity is inhibited . This mechanism of regulation can be explained as follows-

  • High ATP/Low ADP concentration
  • Less ADP available for phosphorylation
  • Lower rate of oxidative phosphorylation
  • Decreased rate of electron transport
  • Less availability of oxidized coenzymes
  • TCA cycle enzymes are inhibited.
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Reactions of TCA cycle (Contd.)

TCA Cycle

Figure-1- Reactions of TCA cycle

For  details of reactions 1-4 click the link below

http://www.namrata.co/tca-cycle-lecture-1/

 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 (Figure-2). This reaction is catalyzed by succinyl CoA synthetase (succinate thiokinase).

 

 

 Formation of Succinate

Figure-2- Formation of succinate and GTP

Biological significance

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 (Figure-4) 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.

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

 Reaction catalyzed by succinate dehydrogenase

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

Clinical significance- The enzyme succinate dehydrogenase is inhibited by Malonate, a competitive inhibitor of the enzyme.

b)  Addition of water–  Fumarase (fumarate hydratase) catalyzes the addition of water across the double bond of fumarate, yielding malate (Figure-3)

                       

    Formation of malate       

Figure-3-Formation of Malate from Fumarate

c) Second dehydrogenation reaction -Malate is converted to oxaloacetate by malate dehydrogenase, a reaction requiring NAD+.

 Formation of oxaloacetate

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.

 Formation of oxaloacetate from succinate

Figure-4- The regeneration of Oxaloacetate from Succinate. Dehydrogenation is followed by hydration to be followed by second dehydrogenation to form oxaloacetate.

 Energy yield per Acetyl co A per turn of cycle

 The net reaction of the citric acid cycle is-

 Net reaction of TCA cycle

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.

ATP Formation in the Catabolism of Glucose

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.

Pathway Reaction Catalyzed by Method of ATP Formation ATP per Mol of Glucose
Glycolysis Glyceraldehyde 3-phosphate dehydrogenase Respiratory chain oxidation of 2 NADH 6*
 
Phosphoglycerate kinase Substrate level phosphorylation 2
Pyruvate kinase Substrate level phosphorylation 2
                                                                   Total 10
Consumption of ATP for reactions of hexokinase and phosphofructokinase –2
                                                                   Net 8
Pyruvate dehydrogenase complex Pyruvate dehydrogenase Respiratory chain oxidation of 2 NADH 6
Citric acid cycle Isocitrate dehydrogenase Respiratory chain oxidation of 2 NADH 6
α-Ketoglutarate dehydrogenase Respiratory chain oxidation of 2 NADH 6
Succinate thiokinase Substrate level phosphorylation 2
Succinate dehydrogenase Respiratory chain oxidation of 2 FADH2
 
4
Malate dehydrogenase Respiratory chain oxidation of 2 NADH 6
                                                                   Net 30
Total per mol of glucose under aerobic conditions 38
Total per mol of glucose under anaerobic conditions 2
  •  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. As in brain and skeletal muscle due to transport of reducing equivalents through glycerophosphate shuttle the net yield of ATP molecules per glucose oxidation is 36  in comparison to 38 ATP in other cells of the body  .
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Tricarboxylic acid (TCA) cycle, also called Krebs cycle or 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. 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 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).

Overview of TCA cycle                                        

 Figure- 1-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-1). 

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-1). 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 (Figure-2).

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

 Flow of electrons from Acetyl coA to O2 through ETC

Figure-2 – Oxygen is required for the citric acid cycle indirectly in as much as it is the electron acceptor at the end of the electron-transport chain, necessary to regenerate NAD+ and FAD. Out of a total of 12 ATP molecules 11 are produced by oxidative phosphorylation. The 12 th ATP is formed by substrate level phosphorylation.

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 TCA cycle (Figure-3)

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.

 TCA Cycle

Figure-3- Reactions of TCA Cycle

Details of reactions

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.(Figure-4) . This reaction, 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.

 Formation of citrate

Figure-4- 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-5) .

 Formation of isocitrate

Figure-5- 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. 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.It is an example of suicidal inhibition.

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 (Figure-6).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.

 Formation of Alpha keto glutarate

Figure- 6-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-7) .

 Formation of succinyl co A

Figure-7- 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—thiamine pyrophosphate, 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.

 Reactions to be continued in the next post …

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Under anaerobic conditions,  pyruvate is converted into lactic acid or ethanol, depending on the organism. Under aerobic conditions, pyruvate is transported into mitochondria by a proton symporter (Figure-1). In the mitochondrial matrix, pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex to form acetyl CoA.

 Pyruvate dehydrogenase complex catalyzed reaction

 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 symport

Figure-1- pyruvate symport

Components of Pyruvate dehydrogenase complex

1)  Enzymes- The pyruvate dehydrogenase complex is a large, highly integrated complex of 2 types of enzymes-

A)- Catalytic enzymes

a) Pyruvate dehydrogenase (E1)

b) Dihydrolipoyl transacetylase (E2)

c) Dihydrolipoyl dehydrogenase (E3)

B)- Regulatory Enzymes

a) PDH Kinase

b) PDH Phosphatase

The pyruvate dehydrogenase complex consists of a number of polypeptide chains of each of the three component  catalytic enzymes, and the intermediates do not dissociate, but remain bound to the enzymes.

2) Coenzymes of PDH complex

Five coenzymes: thiamine pyrophosphate (TPP), lipoic acid, CoASH, FAD and NAD+ participate in the overall reaction (Figure-2)

Pyruvate dehydrogenase is a member of a family of homologous complexes that includes the citric acid cycle enzyme α- ketoglutarate dehydrogenase, a branched-chain α-ketoacid dehydrogenase, and acetoin dehydrogenase, found in certain prokaryotes. These complexes are large, with molecular masses ranging from 4 to 10 million daltons.

Reaction catalyzed by PDH Complex

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

 Pyruvate to Acetyl co A

Reaction sequence in PDH complex catalyzed reaction

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

Reaction steps

 i) Pyruvate is decarboxylated by the pyruvate dehydrogenase component of the enzyme complex to a hydroxyethyl derivative of the thiazole ring of enzyme-bound thiamine pyrophosphate (Figure-2)

ii) Hydroxyethyl TPP in turn reacts with oxidized lipoamide, the prosthetic group of dihydrolipoyl transacetylase, to form acetyl lipoamide. Thiamine is vitamin B1 and in deficiency, glucose metabolism is impaired, and there is significant (and potentially life-threatening) lactic and pyruvic acidosis.

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

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

v) Finally, the reduced flavoprotein is oxidized by NAD+, which in turn transfers reducing equivalents to the respiratory chain. (Figure-2)

 REACTION STEPS PDH complex

Figure-2- Reactions of PDH complex.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.

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

Covalent modification– It is also regulated by phosphorylation of three serine residues on the pyruvate dehydrogenase component of the multienzyme complex.

PDH exists in two forms-

i) PDH-a form which is active and dephosphorylated form

ii) PDH -b form which is inactive and phosphorylated form

PDH kinase, causes phosphorylation resulting in decreased activity, and

PDH phosphatase causes an increase in activity by dephosphorylation of the enzyme

Regulation of PDH Kinase

Positive effectors– NADH and acetyl-CoA are powerful positive effectors on PDH kinase, the enzyme thus inactivates PDH by converting it to the phosphorylated PDH-b form (Figure-3)

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.

Negative effector

Pyruvate is a potent negative effector on PDH kinase, with the result that when pyruvate levels rise, PDH-a (active form) 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.

Regulation of PDH complex 

Figure -3-Regulation of PDH complex by covalent modification. Activation of PDH kinase causes inactivation of PDH, whereas activation of  PDH phosphatase bring about activation of enzyme

Regulation of PDH phosphatase

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.

Energetics of PDH complex

Two pyruvate molecules are obtained from one glucose molecule through glycolysis. Each of the pyruvate yields one NADH, thus there are two NADH molecules to be oxidized through the electron transport chain.

 Pyruvate dehydrogenase complex catalyzed reaction

Each of NADH yields 3 ATP molecules, thus a total of 6 ATP molecules are produced at the level of PDH complex.

Clinical Significance

PDH Complex deficiency

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.

Pathophysiology

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.

Clinical Manifestations

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

Laboratory Diagnosis

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

Treatment

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.

Prognosis

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

Inhibitors of PDH Complex

Arsenite and mercuric ions react with the —SH groups of lipoic acid and inhibit pyruvate dehydrogenase complex .

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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. The reactions of pyruvate of prime importance are as follows-

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+.  

Alcohol-fermentation

Reaction 1- showing the conversion of pyruvate to Ethanol  The net result of this anaerobic process is:

 

 Reaction-of-alcohol-fermentation

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 (Reaction-1). The ethanol formed in alcoholic fermentation provides a key ingredient for brewing and winemaking.

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 (Reaction-2)  

Lactate-fermentation

Reaction-2- showing the conversion of pyruvate to Lactate The overall reaction in the conversion of glucose into lactate is:  

 

Reaction-of-lactate-fermentation

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 lactate or 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.

Reaction-of-PDH-complex 

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 multienzyme 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.  

Pyruvate-to-oxaloacetate-conversion

Reaction-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.  

Transamination-of-pyruvate

Reaction-4- showing the conversion of Pyruvate to Alanine by transamination. The reaction is catalyzed by ALT (Alanine transferase also called SGPT-Serum glutamate alanine transaminase) This reaction is important for the catabolism and synthesis of nonessential amino acids

6. Malate- Pyruvate can be directly converted to oxaloacetate or it is first carboxylated to malate and then decarboxylated to form oxaloacetate (Figure-1).  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.  Pyruvate-to-malate  

Figure-1-showing the formation of Oxaloacetate from Pyruvate.

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

For details of PDH complex- see the next post.

 

 

 

 

 

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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 (Induction and Repression)

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-1 for step 3, and pyruvate kinase for step 10, are the primary steps for  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 (Induction/repression). Insulin up regulates (induces) the transcription of Glucokinase, phosphofructo kinase-1,  and pyruvate kinase, while glucagon down regulates(represses) 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.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.
  • Glucokinase is an inducible enzyme—the amount present in the liver is controlled by insulin. 

2) Regulation of Phospho fructokinase-1

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 (its substrate) 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

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-1.
  • Phosphofructokinase-1 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-1 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-1 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-1 and, by inhibiting fructose-1,6-bisphosphatase, the enzyme that catalyzes this reaction in the opposite direction.
  • Fructose 2,6 bisphosphate is produced from fructose-6-P by Phosphofructokinase-2 enzyme (an isomer of PFK-1)- see the details below-

d) Effect of pH

  • A fall in pH also inhibits Phosphofructokinase-1 activity.
  • The inhibition of phosphofructokinase-1 by H+ prevents excessive formation of lactic acid and a precipitous drop in blood pH (acidosis).

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 or it can be used in the Uronic acid pathway depending upon the cellular requirement.

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-1 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

a) Allosteric modification

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

b) Covalent modification

Liver pyruvate kinase is regulated by covalent modification. Hormones such as glucagon activate a c-AMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme (Figure-1). The phosphorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher Km for phosphoenol pyruvate ( 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. This hormone-triggered phosphorylation, prevents the liver from consuming glucose when it is more urgently needed by brain and muscles.

In the well fed state, under the influence of Insulin the dephosphorylated form predominates, the enzyme remains active and the glucose utilization is promoted so as to maintain the blood glucose concentration within the physiological range.

 Regulation of pyruvate kinase by covalent modification

Figure –1-  Regulation of pyruvate kinase by allosteric effectors and by covalent modification. The enzyme becomes active in the low energy state (excess of AMP), while it becomes inactive in the energy rich state(high ATP concentration ). Alanine is produced from pyruvate by transamination, its high concentration also signifies excess pyruvate/ product of glycolysis, hence the pathway is switched off in conditions of excess alanine. Similarly excess Acetyl co A produced from pyruvate also inhibits this enzyme by allosteric modification.In the presence of high glucose concentration the enzyme remains in the dephosphorylated or active form whereas in conditions of starvation/low glucose concentration, the enzyme remains in the phosphorylated or inactive form to inhibit glycolysis.

4) Effect of hypoxia

Hypoxia stimulates glycolysis, by stimulating the peripheral uptake of glucose by increasing the number of  glucose transporters and also by increasing the activities of glycolytic enzymes. 

5) Role of hormones

Insulin promotes glycolysis by

  • Inducing the regulatory enzymes (Glucokinase, PFK-1 and Pyruvate kinase)
  • Covalently modifying  pyruvate kinase enzyme (maintaining it in the dephosphorylated form)

Glucagon inhibits glycolysis by

  • Repressing the key regulatory enzymes
  • Covalently modifying  pyruvate kinase enzyme (maintaining it in the phosphorylated form)

Role of fructose 2,6 bisphosphate

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 its synthesis (phosphorylating fructose 6-phosphate) and degradation (dephosphorylating fructose 2,6- bisphosphate).

Synthesis of fructose-2,6-bisphosphate

Fructose 2,6-bisphosphate is formed in a reaction catalyzed by phosphofructokinase- 2 (PFK2), a different enzyme from phosphofructokinase-1.

Degradation of Fructose 2,6-bisphosphate

Fructose 2,6-bisphosphate is hydrolyzed to fructose 6-phosphate by a specific phosphatase, fructose2,6 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 55kd polypeptide chain. This bifunctional enzyme contains an N-terminal regulatory domain, followed by a kinase domain and a phosphatase domain (Figure-2). The bifunctional enzyme itself probably arose by the fusion of genes encoding the kinase and phosphatase domains.

 Bifunctional PFK-2 and Fructose 2,6 bisphosphatase enzyme

Figure –2- 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 Fr 2,6-BP (Figure-3). Hence, an abundance of fructose 6phosphate leads to a higher concentration of Fr2,6BP, which in turn stimulates phosphofructokinase-1. 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-3). 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.

 

 Regulation of concentration of fructose 2,6 bisphosphate

Figure- 3- In the bifunctional enzyme, upon phosphorylation by c-AMP mediated cascade, the bisphosphatase enzyme becomes active while PFK-2 becomes inactive, the net concentration of fructose 2,6 bisphosphate decreases, glycolysis is inhibited. Dephosphorylation brings about the opposite effects, PFK-2 activity is increased while bisphosphatase activity is decreased, the net concentration of fructose 2,6 bisphosphate is increased ,  PFK-1 activity is stimulated and  rate of glycolysis is also increased.

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.

Summary of regulation of glycolysis

Sr. No. Features Hexokinase Glucokinase Phosphofructokinase-1 Pyruvate kinase
1) Induction/Repression Non inducible Inducible-Induced by insulin, repressed by Glucagon Inducible-Induced by insulin , repressed by Glucagon Inducible-Induced by insulin, repressed by Glucagon
2) Effect of substrate Activity increases in the presence of excess glucose Active only in the presence of large concentration of glucose due to high Km Activity increases with the increasing concentration of glucose Activity increases with the increasing concentration of glucose
3) Feed back inhibition Inhibited by Glucose-6-P Not inhibited Not inhibited unless there is a block at some enzymatic level as in pyruvate kinase deficiency Inhibited by increasing concentration of pyruvate
4) Allosteric modification ATP and Citrate are negative modifier whereas AMP and fructose 2,6 bisphosphate are positive modifiers ATP, Acetyl coA and Alanine are negative modifiers while AMP and fructose 1,6 bisphosphate are positive modifiers.
5) Covalent modification Active in the dephosphorylated form while inactive in the Phosphorylated form
6) Role of hormones Insulin stimulates,Glucagon inhibits Insulin stimulates,Glucagon inhibits Insulin stimulates,Glucagon inhibits Insulin stimulates,Glucagon inhibits
7) Effect of  hypoxia Activity increases Activity increases Activity increases Activity increases

 

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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 (Figure-1).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.

 Fate of NADH under anerobic conditions

Figure-1- 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:

 Summary of glycolysis

Pathway Reaction Catalyzed by Method of ATP Formation ATP per Mol of Glucose
Glycolysis Glyceraldehyde 3-phosphate dehydrogenase Respiratory chain oxidation of 2 NADH 6
 
Phosphoglycerate kinase Substrate level phosphorylation 2
Pyruvate kinase Substrate level phosphorylation 2
   Total 10
Consumption of ATP for reactions of hexokinase and phosphofructo kinase -1 reactions –2
    Net 8

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.

Biological Significance of Glycolysis

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.

 To See the significance of 2,3 BPG, follow the link as under

http://www.namrata.co/glycolysis-lecture-2/

Clinical Significance of Glycolysis

Pyruvate kinase deficiency

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.

For details of pyruvate kinase deficiency, follow the link as under

http://www.namrata.co/pyruvate-kinase-deficiency-a-case-study-and-complete-discussion/

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Glycolysis- Reactions (Contd.)

For reaction -1- Read the previous post.

http://www.namrata.co/glycolysis-lecture-1/

Reaction 2: Conversion of Glucose-6-P to Fructose-6-Phosphate

  • The second step in glycolysis is a common type of metabolic reaction: the isomerization of a sugar.
  • 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-1).
  • 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: The Second Priming Reaction- Conversion of Fructose-6-Phosphate to Fr-1,6 bisphosphate

  • The next step in the glycolytic pathway is the phosphorylation of fructose by phosphofructo kinase (Figure-1) to form Fr 1,6 bisphosphate.
  • 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.
  • The substrate that provides the phosphoryl group is ATP.
  • The large free energy change of the reaction makes it a likely candidate for regulation,
  • The phosphofructo kinase reaction is an important site of regulation—indeed, the most important site in the glycolytic pathway. Phospho fructokinase is the “valve” controlling the rate of glycolysis.

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

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

Reaction 5: Triose Phosphate Isomerase catalyzes isomerization of trioses

  • 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 (Figure-1) .
  • 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.

Clinical Significance

Bromo- hydroxy acetone phosphate, a structural analog of Dihydroxy acetone phosphate acts as a competitive inhibitor of phospho triose isomerase ,  glycolysis is turned off in its presence.

 

 Reactions of glycolysis

 Figure- 1-Reactions of Glycolysis

The Second Phase of Glycolysis (Pay off phase)

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

Reaction 6:  Conversion of Glyceraldehyde-3-Phosphate to 1,3 bisphosphoglycerate

The reaction is catalyzed by Glyceraldehyde-3-Phosphate Dehydrogenase

Two processes are involved at this step

a) Oxidation by dehydrogenation-  Glyceraldehyde-3-phosphate is oxidized to 1,3-bisphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase (Figure-1).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.

b) Phosphorylation- Phosphorylation of the substrate (Figure-1) takes place by inorganic phosphate, ATP is not the phosphate donor. The product is a high energy compound.

Biological significance-Each NADH produced as a result of this reaction upon oxidation in the electron transport chain yields 3 ATPs, thus a total of 6 ATP molecules are produced at this step under aerobic conditions. But in cells lacking mitochondria and under anaerobic conditions, no ATP molecules are produced at this step.

Clinical significance-1)The enzyme can be inactivated by reaction with iodoacetate, which reacts with and blocks the essential cysteine sulfhydryl.

2) The glyceraldehyde-3-phosphate dehydrogenase reaction is the site of action of arsenate (AsO43-) , an anion analogous to phosphate. Arsenate is an effective substrate in this reaction, forming 1-arseno-3-phosphoglycerate (Figure-2) 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.

 1 Arseno, 3 phospho glycerate

Figure-2- 1- Arseno-3-phosphoglycerate is an unstable compound.

Reaction 7 : Phosphoglycerate Kinase (Conversion of 1,3 bisphosphoglycerate to 3,Phosphoglycerate)

The enzyme phosphoglycerate kinase transfers a phosphoryl group from 1,3-bisphosphoglycerate to ADP to form an ATP (Figure-1). 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.

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.

Biological significance

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-3). 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 .

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 conversion of 1,3-BPG to 2,3-BPG, which enhances the deoxygenation of hemoglobin. Low pH inhibits the activity of bisphosphoglycerate mutase and activates bisphosphoglycerate phosphatase, which favors generation of ATP.

 RL Shunt

Figure -3- RL Shunt- 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.

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-4)

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, anemia 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.

 Effect of 2,3 BPG

Figure-4- Binding of 2,3 BPG to hemoglobin reduces affinity of Hb for oxygen

c) 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.

Reaction 8:  Conversion of 3- Phosphoglycerate to 2-Phosphoglycerate by 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-1), 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-1). The reaction in essence involves a dehydration—the removal of a water molecule—to form the enol structure of PEP. Phosphoenol pyruvate is a high energy compound.

Clinical significance

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. 

A mixture of sodium fluoride and potassium oxalate is used for sample collection for blood glucose estimation. Potassium oxalate acts as an anticoagulant while sodium fluoride inhibits glycolysis. If the mixture is not used, glucose can be oxidized anaerobically and falsely low values of blood glucose can be obtained.

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 (Figure-1). 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.

 This is the second place for substrate level phosphorylation in Glycolysis.

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Glycolysis is the stepwise degradation of glucose (and other simple sugars).

 Salient features of Glycolysis

  • It is 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 and the cells 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.
  • The other hexoses, trioses and glycerol can also be oxidized through this pathway.

Overview of Glycolysis

Glycolysis consists of two phases. In the first phase, through a series of five reactions, glucose is broken down to two molecules of glyceraldehyde-3-phosphate. Phase 1 consumes two molecules of ATP. It is also called ,’Energy investment phase”.

In the second phase, five subsequent reactions convert these two molecules of glyceraldehyde-3-phosphate into two molecules of pyruvate (Figure-1). The later stages of glycolysis result in the production of 2 molecules of ATP (in the absence of O2)  and 8 ATP molecules (in the presence of O2) per molecule of glucose oxidized. Phase 2 is also called ” Pay back phase or phase of energy generation “(Figure-2) .

 Overview of glycolysis

Figure-1- Phase-1 of Glycolysis involves two phosphorylation reactions to form Hexose bisphosphate ( Fructose-1,6 bisphosphate) that undergoes lysis to form two phosphorylated trioses which are subsequently converted to two pyruvate molecules by phase 2 reactions.

 Phases of glycolysis

Figure-2- Phase -1 involves consumption of two ATP molecules to energize the substrate for easy cleavage. The ATP debt is paid back in the 2 nd phase by net generation of two ATP molecules under anaerobic and 8ATP molecules per glucose under aerobic conditions. Each NADH upon oxidation in electron transport chain yields 3 ATP molecules.

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.

 Reactions of glycolysis

Figure-3- Out of the 10 reactions, 3 reactions are irreversible and the seven reversible reactions are of advantage in glucose production in the pathway of gluconeogenesis.

Details of reactions

Phase-1 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 (Figure-2) .

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).

Reaction-1 of glycolysis

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.

The incorporation of a phosphate into glucose in this energetically favorable reaction is important for several reasons-

1) 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-4).

2) Moreover, rapid conversion of glucose to glucose-6-phosphate keeps the intracellular concentration of glucose low, favoring diffusion of glucose into the cell.

3) In addition the addition of the phosphoryl group begins to destabilize glucose, thus facilitating its further metabolism.

4) Furthermore, 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.

 Glucose trapping

Figure-4- Glucose is trapped inside the cell by phosphorylation to from 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.

The important differences between hexokinase and glucokinase can be summarized as follows-

S.No. Features Hexokinase Glucokinase
1) Tissue distribution Most tissues Liver and β cells of Pancreas
2) Km Low (0.05 mM) High (10 mM)
3) Vmax   Low High
4) Product inhibition Inhibited by G6P Not inhibited by G6P
5) Inducible Non inducible Inducible
6) Function/Biological significance Maintains intracellular glucose concentration Maintains blood glucose concentration
7) Clinical Significance Deficiency causes hemolytic anemia Decreased activity is observed in diabetes mellitus

Significance of Glucose-6-phosphate

Glucose 6-phosphate is an important compound at the junction of several metabolic pathways: glycolysis, gluconeogenesis, the pentose phosphate pathway, uronic acid pathway, glycogenesis, and glycogenolysis. It can enter any of the pathways depending upon the cellular requirement.

Reactions to be continued …….

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Enzyme Pathway Effect of substrate concentration Allosteric modification/ Feedback Inhibition Induction/

Repression

Hexokinase Glycolysis   Feedback inhibition by Glucose-6-P
Glucokinase Glycolysis Stimulated by high carbohydrate diet 

Activity decreased during fasting

Induced by Insulin

Repressed by-

 Glucagon

Phospho

fructokinase-1

Glycolysis Stimulated by high carbohydrate diet 

Activity decreased during fasting

Activators-5’AMP,

fructose 6-phosphate,

fructose 2,6-bisphosphate

Inhibitors

Citrate, ATP,

Induced by Insulin

Repressed by

 Glucagon

Pyruvate Kinase Glycolysis Stimulated by high carbohydrate diet 

Activity decreased during fasting

Activators-Fructose 1,6-bisphosphate,

insulin

 

Inhibitors

ATP,

alanine,

glucagon

Induced by Insulin 

Repressed by Glucagon

Pyruvate dehydrogenase complex Pyruvate to Acetyl Co A Stimulated by high carbohydrate diet 

Activity decreased during fasting

Activators-CoA, NAD+,

insulin,

ADP,

Pyruvate

 

Inhibitors

Acetyl CoA,

NADH,

ATP (fatty acids, ketone bodies)

 
Pyruvate carboxylase Gluconeogenesis Inhibited by high carbohydrate diet 

Stimulated during fasting

 Activator-Acetyl CoA

Inhibitor

ADP

 Induced by Glucocor-ticoids, glucagon, 

epinephrine

 

Repressed by

Insulin

Fructose 1,6 bisphosphatase Gluconeogenesis Inhibited by high carbohydrate diet 

Stimulated during fasting

Activator-Citrate

 

Inhibitor

AMP,

Fr 2,6 bisphosphate

Induced by 

Glucocortic-oids,

 glucagon,

epinephrine

 

Repressed by

Insulin

Glucose-6-P dehydrogenase HMP pathway Stimulated by high carbohydrate diet 

Activity decreased during fasting

Activator-NADP+ Induced by Insulin
Glycogen

Synthase

Glycogenesis Stimulated by high carbohydrate diet 

Activity decreased during fasting

Activators-Glucose and Glucose-6-P

and Insulin (By causing dephosphorylation)

inhibitors

Glucagon(By c AMP mediated phosphorylation)

 

 
Glycogen

Phosphorylase

Glycogenolysis Stimulated during fasting Inhibited in the fed state Activators-Ca ++,

Low Glucose concentration,

Glucagon (By c AMP mediated phosphorylation)

Inhibitor-

Insulin (By causing dephosphorylation)

 

 

 

 

 

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Type Name Enzyme Deficiency Clinical features
0 Glycogen synthase Hypoglycemia; hyperketonemia; early death
I Von Gierek’s disease Glucose 6-phosphatase Glycogen accumulation in liver and renal tubule cells; hypoglycemia; lactic acidemia; ketosis; hyperlipidemia
II Pompe’s disease Lysosomal α1->4 and

α 1->6 Glucosidase (acid maltase)

Accumulation of glycogen in lysosomes: juvenile onset  variant, muscle hypotonia, death from heart failure by age 2; adult onset variant, muscle dystrophy
III Limit dextrinosis, Forbe’s or Cori’s disease Debranching enzyme Fasting hypoglycemia; hepatomegaly in infancy; accumulation of characteristic branched polysaccharide 
IV Amylopectinosis, Andersen’s disease Branching enzyme Hepatosplenomegaly; accumulation of polysaccharide with few branch points; death from heart or liver failure in first year of life
V Myo phosphorylase deficiency, McArdle’s syndrome Muscle phosphorylase Poor exercise tolerance; muscle glycogen abnormally high (2.5–4%); blood lactate very low after exercise
VI Hers’ disease  Liver phosphorylase Hepatomegaly; accumulation of glycogen in liver; mild hypoglycemia; generally good prognosis
VII Tarui’s disease Muscle and erythrocyte Phospho fructokinase 1 Poor exercise tolerance; muscle glycogen abnormally high (2.5–4%); blood lactate very low after exercise; also haemolytic anemia
VIII Liver phosphorylase kinase Hepatomegaly; accumulation of glycogen in liver; mild hypoglycemia; generally good prognosis.
IX Liver and muscle phosphorylase kinase Hepatomegaly; accumulation of glycogen in liver and muscle; mild hypoglycemia; generally good prognosis
X cAMP-dependent protein kinase A Hepatomegaly; accumulation of glycogen in liver

 

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S.N. Disease Biochemical Defect and Pathogenesis Inheritance and classification Clinical manifestations Lab.Investigations Treatment
1. Lactose Intolerance Deficiency of lactase. Lactose present in ingested dairy products remains uncleaved and passes intact into the colon. The accumulated lactose produces osmotic effects and undergoes fermentation to produce a variety of abdominal manifestations. There are three major types of lactose intolerance:1) Primary lactose intolerance- begins after about age 2   

2) Secondary lactose intolerance occurs because of mucosal damage or from medications resulting from certain gastrointestinal diseases,

3) Congenital lactase deficiency  present at birth, Autosomal recessive inheritance

 

 

 

 

 

 

 

Common symptoms include – abdominal pain, bloating, gas, diarrhea, nausea.  1. Hydrogen Breath Test – Normally, very little hydrogen is detectable in the breath, but undigested lactose produces high levels of hydrogen. 2)Stool Acidity Test- Undigested lactose creates lactic acid and other fatty acids that can be detected in a stool sample.

Glucose may also be present in the stool as a result of undigested lactose.

 

 

 

 

Lactose-free, lactose-reduced milk, Soy milk. Lactase enzyme drops or tablets

Getting enough calcium is important

2. Pyruvate kinase deficiency (PKD)  An erythrocyte enzymopathy involving Glycolysis. A discrepancy between erythrocyte energy requirements and ATP-generating capacity produces irreversible membrane injury, resulting in cellular distortion, rigidity, and dehydration. This leads to premature erythrocyte destruction by the spleen and liver with the resultant anemia. Autosomal recessive  Severe anemia, jaundice, Kernicteruschronic leg ulcers,

Growth failure and failure to thrive

Hb, Serum Bilirubin and confirmation by enzyme assays Mild to moderate disease only supportive treatment. Severe case – blood transfusion or bone marrow transplantation can be done.

Splenectomy is indicated only in severe anemia.

3. PDH- complex deficiency Pyruvate dehydrogenase complex (PDC) converts pyruvate to acetyl-coenzyme A (CoA), 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.

Malfunction of this cycle deprives the body of energy.

An abnormal lactate buildup results in nonspecific symptoms.

X-linked Severe lethargy, poor feeding, tachypnea), especially during times of illness, stress, or high carbohydrate intake. Progressive neurological symptoms like developmental delay, intermittent ataxia, poor muscle tone, abnormal eye movements, or seizures are present in early infancy.

 

Serum lactate and pyruvate levels,Urinary and serum Alanine level.

MRI for detection of neurological damage

.

Symptomatic treatment. -Low carbohydrate diet Thiamine supplementation, –  

Ketogenic diet and oral citrate to correct acidosis.

4.  Glucose-6-phosphatase dehydrogenase (G6PD) deficiency Glucose 6-phosphate dehydrogenase (G6PD) is an enzyme critical in the redox metabolism of all aerobic cells .In red cells, its role is even more critical because it is the only source of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which, directly and via reduced glutathione (GSH), defends these cells against oxidative stress. With the help of the enzyme glutathione peroxidase, reduced glutathione converts harmful hydrogen peroxide to water. Deficiency leads to haemolytic anemia

 

X-linked disorder The vast majority of people remain asymptomatic throughout life other develop haemolytic anemia in response to oxidative challenges. Acute haemolytic anemia develops in response to  ingestion of fava beans, drugs and infections.

Typically, a haemolytic attack starts with malaise, weakness, and abdominal or lumbar pain.

After an interval of several hours to 2–3 days, the patient develops jaundice and often dark urine, due to hemoglobinuria.

The onset can be extremely abrupt, especially with favism in children.

Hb low-The anemia is moderate to extremely severe, usually normocytic and normochromic associated with hemoglobinuria,LDH is high and so is the unconjugated bilirubin,

The confirmation is done by measuring the actual enzyme activity.

Identification and discontinuation of the precipitating agent Oxygen and bed rest, which may afford symptomatic relief.

Prevention of drug-induced hemolysis is possible in most cases by choosing alternative drugs.

 

5. Hereditary Galactosemia Galactose-1-phosphate uridyl Transferase (GALT) deficiency is the most common enzyme deficiency that causes Galactosemia. The other enzyme deficiencies which can produce Galactosemia are- Galactokinase and Uridine diphosphate (UDP) galactose-4-epimerase enzyme deficiencies. Autosomal recessive. The cardinal features of classic galactosemia are hepatomegaly, cataracts, and mental retardation.Poor growth, liver dysfunction and jaundice are also present. 1. Urine test for reducing sugar is positive. The confirmation is done by thin layer chromatography.2.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.

3.Serum Bilirubin and Transaminases  may be high.

4-Confirmation is by enzyme estimation in erythrocytes.

Galactose free diet is  recommended to prevent the accumulation of galactose
6. Hereditary fructose intolerance Hereditary fructose intolerance is caused by mutation in the gene encoding Aldolase B enzyme. The rate of phosphorylation  by fructokinase is very high and in the presence of Aldolase B deficiency

ATP depletion takes place which is manifested in the form of hyperuricemia, lacticacidemia and liver dysfunction.

An autosomal recessive trait These patients are healthy and asymptomatic until fructose or sucrose (table sugar) is ingested (usually from fruit, sweetened cereal, or sucrose-containing formula).Clinical features include recurrent abdominal pain, vomiting and hypoglycemia.

Long term exposure leads to liver and kidney failure.

 

Urine test is positive for reducing sugar, aminoaciduria, proteinuria and phosphaturia may also be associatedLiver and kidney function tests may be abnormal.

The confirmation is done by enzyme estimation.

Total avoidance of fructose containing foods.
7. Essential Pentosuria Deficiency of Xylitol dehydrogenase enzyme. This enzyme converts L Xylulose to Xylitol.

Failure to convert leads to accumulation of

L Xylulose in blood and is also excreted excessively in urine

Autosomal recessive Asymptomatic. Just a chance finding. Urine test is positive for reducing sugar.Confirmation by Bial’s test and loading test by

D-Glucuronic acid.

No treatment is needed

 

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This is an enzyme of Uronic acid pathway. 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, some endogenous compounds like bilirubin,certain hormones, metabolites 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.(Figure-1)

 

 

Figure1-showing the overview of Uronic acid pathway

 

Biological significance of UDP –Glucuronyl Transferase

UDP glucuronate the active form of Glucuronic acid, can readily donate the Glucuronic acid component under the catalytic activity of UDP –Glucuronyl Transferase for the following functions-

1)   Detoxification of foreign compounds and drugs- During detoxification, the glucuronate residues are covalently attached to lipid soluble 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. UDP-Glucuronic acid is the Glucuronyl donor, and a variety of glucuronosyl transferases, present in both the endoplasmic reticulum and cytosol, are the catalysts. Molecules such as 2-acetylaminofluorene (a carcinogen), aniline,benzoic acid, meprobamate (a tranquilizer), phenol, and many steroids are excreted as glucuronides. The glucuronide may be attached to oxygen, nitrogen,or sulfur groups of the substrates. Glucuronidation is probably the most frequent conjugation reaction.

2)   Synthesis of Mucopolysaccharides-UDP Glucuronic acid is an essential component of Hyaluronic acid and heparin.

3)   Conjugation of Bilirubin-Bilirubin is nonpolar and would persist in cells (eg, bound to lipids) if not rendered water-soluble. Hepatocytes convert bilirubin to a polar form, which is readily excreted in the bile, by adding Glucuronic acid molecules to it. This process is called conjugation.

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 glucuronosyl donor, and is referred to as bilirubin-UGT. BilirubinMonoglucuronide is an intermediate and is subsequently converted to the diglucuronide. Most of the bilirubin excreted in the bile of mammals is in the form of bilirubin diglucuronide.(Figure-2)

 

 

Figure- 2-showing the conjugation of bilirubin

Clinical significance Diminished activity of Bilirubin UDP Glucuronyl Transferase (UGT)

1) Neonatal “Physiologic Jaundice”

This transient condition is the most common cause of Unconjugated hyper-bilirubinemia. It results from an accelerated hemolysis around the time of birth and an immature hepatic system for the uptake, conjugation, and secretion of bilirubin. Not only is the bilirubin-UGT activity reduced, but there probably is reduced synthesis of the substrate for that enzyme,UDP-glucuronic acid. Since the increased amount of bilirubin is unconjugated,it is capable of penetrating the blood-brain barrier when its concentration in plasma exceeds that which can be tightly bound by albumin (20–25 mg/dL). This can result in a hyperbilirubinemic toxic encephalopathy, or kernicterus,which can cause mental retardation. Because of the recognized inducibility of this bilirubin UGT enzyme system, phenobarbital has been administered to jaundiced neonates and is effective in this disorder. In addition, exposure to blue light (phototherapy) promotes the hepatic excretion of unconjugated bilirubin by converting some of the bilirubin to other derivatives such as maleimide fragments and geometric isomers that are excreted in the bile.

2) Crigler-Najjar Syndrome, Type I; Congenital Nonhemolytic Jaundice

a) TypeI Crigler-Najjar syndrome -is a rare autosomal recessive disorder. It is characterized by severe congenital jaundice(serum bilirubin usually exceeds 20 mg/dL) due to mutations in the gene encoding bilirubin-UGT activity in hepatic tissues. The disease is often fatal within the first 15 months of life. Children with this condition have been treated with phototherapy, resulting in some reduction in plasma bilirubinlevels. Phenobarbital has no effect on the formation of bilirubin glucuronides in patients with type I Crigler-Najjar syndrome. A liver transplant may be curative. It should be noted that the gene encoding human bilirubin-UGT is part of a large UGT gene complex situated on chromosome 2. Many different substrates are subjected to glucuronosylation, so many glucuronosyl transferases are required.

b) Crigler-Najjar Syndrome Type II-This rare inherited disorder also results from mutations in the gene encoding bilirubin-UGT, but some activity of the enzyme is retained and the condition has a more benign course than type I. Serum bilirubin concentrations usually do not exceed 20 mg/dL. Patients with this condition can respond to treatment with large doses of phenobarbital.

3) Gilbert Syndrome

Again,this relatively prevalent condition is caused by mutations in the gene encoding bilirubin-UGT. It is more common among males. Approximately 30% of the enzyme’s activity is preserved and the condition is entirely harmless.

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Transketolase- is an enzyme of Pentose phosphate pathway.

The pentose phosphate pathway (also called Phospho Gluconate pathway or hexose mono phosphate 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.

This pathway consists of two phases: the oxidative generation of NADPH and the non oxidative 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)

Transketolase transfers the two-carbon unit comprising carbons 1 and 2 of a ketose on to the aldehyde carbon of an aldosesugar. 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 thiamine diphosphate (vitamin B1)as coenzyme. The two-carbon moiety transferred is probably glycoaldehyde boundto thiamine 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- 2)

 

Figure 2- showing the reaction catalyzedby 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 Erythrose4-phosphate. (Figure-3)

 

Figure 3-showing the reaction catalyzed by Transaldolase

In a further reaction catalyzed by Transketolase, Xylulose5-phosphate serves as a donor of glycoaldehyde. In this case Erythrose4-phosphate is the acceptor, and the products of the reaction are fructose6-phosphate and glyceraldehyde 3-phosphate. (Figure 4)

 

 

 

 

 

Figure 4- showing the rearrangement of sugars to form glycolytic intermediates catalyzed by Transketolase

Clinical Significance– Since Transketolase requires the presence of Thiamine pyro phosphate(TPP) as a coenzyme, in the deficiency of TPP, Transketolase activity is grossly affected. Measurement of red blood cell Transketolase activity is an index for the determination of underlying Thiamine deficiency.


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