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

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

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

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

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

 

 

 

 

 

 

 

 

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

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

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

 

 

 

 

 

 

 

 

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

 

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

 Answer-

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

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

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

 

 

 

 

 

 

 

 

 

 

 

 

 

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

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

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

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

                                         Hexokinase                                    Glucokinase

Tissue distribution: Most tissues                                 Liver and β cells of Pancreas

Km                                 Low (0.05 mM)                             High (10 mM)

Vmax                            Low                                                     High

Inhibition by G6P    Yes                                                      No

Inducible                     No                                                       Yes

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

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

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

 

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

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

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

Overview of Glycolysis

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-4- showing  an overview of Glycolysis

Reactions of Glycolysis

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

The First Phase of Glycolysis

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

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

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

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

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

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

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

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

Reaction 3: Phospho fructokinase —The Second Priming Reaction

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

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

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

Reaction 5: Triose Phosphate Isomerase

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

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure-5- Showing reactions of Glycolysis

The Second Phase of Glycolysis

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

Reaction 6: Glyceraldehyde-3-Phosphate Dehydrogenase

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

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

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

 

 

 

 

 

 

 

 

 

 

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

 Reaction 7 : Phosphoglycerate Kinase

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

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

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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

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

Reaction 8: Phosphoglycerate Mutase

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

Reaction 9: Enolase

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

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

Reaction 10: Pyruvate Kinase

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

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

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

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

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

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

 

 

 

 

 

 

 

 

 

 

Figure–8 showing coupling of reactions

Tissues That Function under Hypoxic Conditions Produce Lactate

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

Energy yield per molecule of Glucose oxidized through Glycolysis

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


 

 

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

 

 

 

 

 

 

 

 

 

 

 

 

 

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

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

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

1. Availability of substrate

2. Concentration of enzymes responsible for rate-limiting steps

3. Allosteric regulation of enzymes

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

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

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

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

1) Regulation at the level of Hexokinase and Glucokinase-

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

2) Regulation of Phospho fructokinase

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

a) Role of ATP

 ATP is an allosteric inhibitor of this enzyme.

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

b) Role of Citrate

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

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

c) Role of Fr 2,6 bisphosphate

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

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

3) Regulation of pyruvate Kinase

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

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

 

 

 

 

 

 

 

 

 

 

 

 

 

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

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

Answer- The following effects are seen –

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

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

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

 

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

Or

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

Or

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

Answer-

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

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

Synthesis of fructose2,6-bisphosphate

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

Degradation of Fructose 2,6-bisphosphate

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

Regulation of concentration of Fructose 2,6-bisphosphate

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

 

 

 

 

 

 

 

 

 

 

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

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

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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

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

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

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

Or

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

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

Luebering-Rapapport pathway

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


 

 

 

 

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

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

 Significance of 2,3-bisphosphoglycerate

a) Unloading of Oxygen-

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

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

b) Effect of Hypoxia-

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

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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

c) Smoking and 2,3 BPG

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

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

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

 

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

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

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

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

 

 

 

 

 

 

 

 

Figure-16- showing the Enolase catalyzed reaction

 

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

 

 

 

 

 

 

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

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

(c) An inhibitor of pyruvate kinase-

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

 

 

 

 

 

 

 

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

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

 

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

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

 

 

 

 

 

 

 

 

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

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

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

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

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

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

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

Or

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

Answer-

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

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

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

 

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

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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

 

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

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

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

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

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

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

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

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

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

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

 

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

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

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

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

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

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