The following points highlight the top three pathways of glucose dissimilation in organisms. The pathways are: 1. Embden-Meyerhof Pathway or Glycolysis 2. Pentose Phosphate Cycle (PPC) 3. Entner-Doudoroff Pathway (EDP).
Glucose Dissimilation: Pathway # 1. Embden-Meyerhof Pathway or Glycolysis:
This catabolic pathway leads to splitting of glucose to two molecules of pyruvic acid i.e. a six- carbon compound is broken down to two molecules of three-carbon compounds. This transformation occurs in several steps and, in the process two molecules of ATP are generated by substrate-level phosphorylation. Altogether 10 different enzymes are involved which are present in soluble state in the cytosol as free entities.
The steps of glycolysis and the names of the enzymes are given below in Fig. 8.43:
The details of the individual steps of the glycolytic pathway are discussed:
In the first step a neutral glucose molecule is activated to negatively charged glucose-6-phosphate by the enzyme hexokinase using ATP as the phosphate donor and Mg++ as cofactor. The hexokinase reaction is strongly exergonic and is not freely reversible. The reverse reaction regenerating glucose from glucose 6-phosphate is catalysed by a different enzyme glucose 6-phosphatase which liberates inorganic phosphate.
In the third step, fructose 6-phosphate is further phosphorylated to fructose diphosphate (correctly it should be called fructose bisphosphate). The reaction is catalysed by fructose 6-phosphate kinase also known as phosphofructokinase. It requires ATP and Mg++ and drives the reaction unidirectionally. The reverse reaction (FBP —> F 6-P) is catalysed by di-phospho-fructose phosphatase.
The fourth step consists of cleavage of FBP into two triose phosphates, GAP and DHAP, catalysed by a lyase, called FBP aldolase. The aldolase reaction is reversible and the reverse reaction consists of an aldol condensation. Hence the enzyme is known as aldolase. The cleavage reaction distributes carbon atoms 1, 2 and 3 of FBP to GAP and carbon atoms 4, 5 and 6 to DHAP.
The two triose phosphates are freely inter-convertible through the action of an isomerase, triose phosphate isomerase, but the equilibrium is strongly in favour of DHAP (indicated by a bold arrow), so that, at any given time, about 90% of the triose phosphates is in the form of DHAP. Further breakdown takes place only from GAP. As more and more of GAP is consumed in the subsequent steps of glycolysis, DHAP is converted to GAP to maintain the supply of GAP.
The fifth step involves a complex reaction in which GAP is oxidized to di-phosphoglyceric acid, NAD acting as the H-acceptor to form NADH2. The reaction is catalysed by the enzyme glyceraldehyde 3-phosphate dehydrogenase which has an active -SH group. The enzyme -SH group binds to the aldehyde group of GAP forming a thioester bond followed by oxidation of the aldehyde group to an acid group, NAD acting as coenzyme.
The energy-rich thioester bond is utilized for addition of another phosphate group and the enzyme is liberated in its original SH-enzyme form to initiate another cycle of catalysis. The product is 1, 3-di-phosphoglyceric acid (DPGA).
The six step consists of transfer of the energy-rich phosphate group of DPGA to DPGA to ADP forming ATP and 3-Phosphoglyceric acid (3-PGA). The reaction is catalysed by the enzyme, phospholglyceric acid kinase. The mode of ATP formation involving transfer of an energy-rich phosphate group to ADP is known as substrate-level phosphorylation.
In the seventh step, 3-PGA is transformed to 2-phosphoglyceric acid (2-PGA) by transfer of the phosphate group to the middle carbon atom. The reaction is catalysed by the enzyme, phosphoglyceromutase. The reaction is freely reversible and requires Mg++.
The eighth step, catalysed by the enzyme, enoloase consists of dehydration of 2-PGA to form phosphoenol pyruvic acid (PEP). As a result of removal of H2O from 2-PGA, the phosphate bond becomes energy-rich. The enzyme requires –MG++ or Mn++ as cofactor and is strongly inhibited by fluoride which binds the co-factors and prevents enolase function.
The ninth and the last step of glycolytic pathway is catalysed by the enzyme pyruvic acid kinase which transfers the energy-rich phosphate group of PEP to ADP forming ATP, thereby effecting the second substrate-level phosphorylation. The reaction is strongly exergonic and is irreversible.
It may be noted that except three reactions of the glycolytic pathway, viz. the first step catalysed by hexokinase, the third step catalysed by phosphofructokinase and the last step catalysed by pyruvic acid kinase, the other reactions are reversible. In all these three irreversible steps, ATP is one of the reactants.
Summing up, the glycolytic pathway results in the cleavage of a glucose molecule first into two trioses which are inter-convertible. The triose is oxidized to glyceric acid and subsequently converted to pyruvic acid. Thus, from each half of glucose, one pyruvic acid is formed i.e. two molecules of pyruvic acids from one molecule of glucose are produced.
There is only one oxidation-reduction step catalysed by glyceraldehyde phosphate dehydrogenase in which NADH2 is formed i.e. two NADH2 are produced per glucose molecule, one from each half. Furthermore, two steps are ATP consuming, viz. conversion of glucose to glucose 6-phosphate and fructose 6-phosphate to fructose bisphosphate catalysed by the respective kinases; and there are two ATP producing steps for each half of glucose-viz. conversion of di-phosphoglyceric acid to phosphoglyceric acid (Step 6) and conversion of phosphoenol pyruvic acid to pyruvic acid (Step 9).
In making a balance-sheet of the inputs and outputs of the glycolytic pathway, it is observed that for every molecule of glucose degraded to two molecules of pyruvic acid, two molecules of ATP are consumed and four molecules of ATP are produced, and two molecules of NAD are reduced to NADH2. Net outputs are, therefore,
Glucose –> 2 Pyruvic acid + 2 ATP + 2 NADH2
In aerobic organisms carrying out oxygen respiration, pyruvic acid is further degraded via tricarboxylic acid cycle (TCA cycle or Krebs’ cycle) where carbon atoms of pyruvic acid are oxidized to CO2 and the H-atom are transferred to specific coenzymes, like NAD and FAD. The hydrogen from these coenzymes are fed into the electron transport chain for generation of ATP and finally transferred to oxygen-producing water. NADH2 is in the process oxidized back to NAD and can be used in the oxidation-reduction step of glycolysis. Thus, a continuous supply of NAD for running glycolysis is assured.
Anaerobic organisms which lack TCA cycle and respiratory chain are unable to regenerate NAD from NADH2 in the way the aerobic organisms do. They have to reduce pyruvic acid to either lactic acid or ethanol with the help of NADH2 which is thereby oxidized to NAD. NAD so produced is then utilized for running glycolysis. Moreover, anaerobic organisms have to depend on substrate-level phosphorylation for generation of ATP.
Glucose Dissimilation: Pathway # 2. Pentose Phosphate Cycle (PPC):
This pathway is also known as hexose monophosphate shunt or phosphogluconate pathway. PPC is not a major pathway of glucose catabolism but it operates in many organisms side by side with EMP. Its main significance lies in production of NADPH2 which is required as a coenzyme for many biosynthetic enzymes.
Another important function is to produce pentose’s, particularly ribose which is an essential part of all nucleic acids. PPC is considered as a cyclic pathway in contrast to EMP, because two of the products, viz. fructose 6-phosphate and glyceraldehyde phosphate can be converted back to the initial compound, glucose 6-phosphate.
An outline of PPC is given in Fig. 8.44:
The PPC starts with phosphorylation of glucose to glucose 6-phosphate catalysed by hexokinase and ATP as in EMP.
At the next step, glucose 6-phosphate is oxidized by the NADP-linked enzyme, glucose 6-phosphate dehydrogenase to form 6-phospho-gluconic acid and NADPH2 via an unstable intermediate, 6-phosphogluconolactone.
6-phophogluconic acid is then converted by a process of oxidation and decarboxylation to ribulose 5-phosphate by enzyme 6-phosphogluconic acid dehydrogenase in which NADP again acts as hydrogen acceptor. With this step the oxidation reactions of the PPC are completed.
The reaction of the rest of the cycle involve transfer of two-carbon and three-carbon units of different sugars by two classes of enzymes – transketolase transferring glycoladehyde (CH2OH-CHO)group and transaldolase transferring dihydroxyacetone (CH2OH.CO.CH2OH) group.
Two reactions catalysed by these enzymes are shown:
The balance-sheet of PPC shows that starting with 6-molecules of glucose 6-phosphate (the common first step between EMP and PPC not being taken into account) the following inputs and outputs are obtained:
6 Glucose 6-phosphate + 12 -> NADP 5 Glucose 6-phosphate + 6CO2 + 12 NADPH2 + Pi.
By eliminating 5 glucose 6-phosphate from either side, one gets
Glucose 6-phosphate + 12 NADP -> 12 NADPH2 + 6 CO2 + Pi
Thus, it is seen that for oxidation of each molecule of glucose 6-phosphate, 12 molecules of NADPH2 are produced. The primary importance of PPC is in production of the reductant NADPH2 in the extra-mitochondrial cytoplasm of the cell. The main use is in numerous biosynthetic oxidation — reduction reaction.
It should be noted that NADPH2 cannot be directly oxidised via electron transport system as NADH2 can be. Trans-hydrogenation of NAD by NADPH2 is possible, but it occurs rarely (NADPH2 —> NADP + NADH2).
Another importance of PPC is the production of a number of useful intermediate products, like erythrose which is required for biosynthesis of aromatic compounds, specially aromatic amino acids. Pentose’s produced in PPC are used for synthesis of nucleic acids. Besides, GAP produced in PPC may be channelized into EMP for generation of energy via TCA cycle and electron transport system.
Some of the oxidation-reduction reactions requiring NADPH2 as H-donor are:
1. Biosynthesis of amino acids. The main part of entry of inorganic NH3 into organic compounds is reductive amination of α-ketoglutaric acid yielding glutamic acid. The latter can then transfer the amino group by transamination producing other amino acids.
The details of the PPC balance-sheet is shown in Fig. 8.45. The abbreviations used are GAP for glyceraldehyde phosphate, G6P for glucose 6-phosphate, PGLA for phosphogluconic acid, Ru5P for ribulose 5-phosphate, Xu5P for xylulose 5-phosphate, S7P for sedoheptulose 7-phosphate, E4P for erythrose 4-phosphate, F6P for fructose 6-phosphate and FBP for fructose bisphosphate.
Glucose Dissimilation: Pathway # 3. Entner-Doudoroff Pathway (EDP):
EDP is found only in some prokaryotic organisms, particularly in Gram-negative bacteria. Some of these bacteria, like Pseudomonas saccharophila and Alcaligenes eutrophus exclusively use this pathway for glucose dissimilation. These organisms evidently lack some of the enzymes of the EMP. Some other organisms, like Pseudomonas aeruginosa employ the EDP for breakdown of the major portion of glucose, while the rest is dissimilated by PPC.
A characteristic intermediate of EDP is 2-keto 3-deoxy 6-phosphogluconic acid (KDPG). Hence, the pathway is also known as KDPG pathway. The key enzyme is KDPG-aldolase. The first two reactions of EDP are the same as in PPC.
Glucose is phosphorylated by hexokinase and ATP to form glucose 6-phosphate which is then dehydrogenated by glucose 6-phosphate dehydrogenase, NADP acting as the H-accepting coenzyme, to form 6-phosphogluconic acid.
The next step is characteristic of the EDP. It consists of dehydration and dehydrogenation of 6-phosphogluconic acid producing 2-keto 3-deoxy 6-phosphogluconic acid (KDPG). The enzyme catalyzing this step is 6-phosphogluconic acid dehydrogenase. This is followed by another characteristic reaction catalysed by KDPG-aldoIase in which KDPG is cleaved into pyruvic acid and glyceraldehyde phosphate (GAP).
Of the two products of KDPG aldolase reaction, pyruvic acid is further broken down through the TCA cycle, while GAP is converted to another molecule of pyruvic acid as it is done in case of EMP via 1, 3-di-phosphoglyceric acid, 3-phosphoglyceric acid, 2-phosphoglyceric acid, phosphoenol pyruvic acid and finally pyruvic acid. So, the net oxidation products of one molecule of glucose via EDP are the same as in EMP viz. two pyruvic acids.
The balance-sheet of EDP is given:
A comparison of EDP with EMP reveals that in both the products of glucose breakdown are two molecules of pyruvic acid per molecule of glucose, but in EMP two molecules of ATP are net gains, while in EDP only one ATP is produced.
In summarizing the three modes of glucose catabolism — EMP, PPC and EDP — it may be mentioned that EMP is the chief pathway found in most eukaryotic and prokaryotic organisms, including animal systems. In most organisms PPC is also used for a part of glucose dissimilation.
For example, in E. coli and Bacillus subtilis, 72% and 74% of the total glucose is catabolized via EMP, while the rest via PPC. Again, E. coli which does not use EDP when growing on glucose as substrate can use EDP when using gluconate as substrate.
EDP operates mostly in some Gram-negative bacteria, like Pseudomonas, Alcaligenes, Rhizobium etc. which do not possess some of the enzymes of EMP, particularly FBP-aldolase. Such organisms employ EDP either exclusively for glucose catabolism or partly e.g. Pseudomonas aeruginosa use EDP for breakdown of 71% glucose and 29% by PPC.