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Glycolysis- Reactions (Contd.)

For reaction -1- Read the previous post.

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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