Glyceraldehyde-3-phosphate dehydrogenase mechanism

Analyses of enzyme reaction rates continued to support the formulations of Henri and Michaelis-Menten and the idea of an enzyme-substrate complex, although the kinetics would still be consistent with adsorption catalysis. Direct evidence for the participation of the enzyme in the catalyzed reaction came from a number of approaches. From the 1930s analysis of the mode of inhibition of thiol enzymes—especially glyceraldehyde-phosphate dehydrogenase—by iodoacetate and heavy metals established that cysteinyl groups within the enzyme were essential for its catalytic function. The mechanism by which the SH group participated in the reaction was finally shown when sufficient quantities of purified G-3-PDH became available (Chapter 4). 

Glyceraldehyde phosphate dehydrogenase probably holds the distinction of being the classic thiol enzyme in the minds of most biochemists . The thiol is believed to be involved in the initial attachment of the aldehyde substrate as a thiohemiacetal. The em me-bound thiohemiacetal is then oxidized by NAD generating an enzyme-bound thioester. In more sophisticated proposals for this mechanism the nicotinamide cofactor interacts with the active centre thiol as a charge transfer type of complex. This facilitates the reaction of the thiol with the carbonyl of the substrate. The thiol addition and the electron transfer to nicotinamide occur... 

FIGURE 19.18 A mechanism for the glycer-aldehyde-3-phosphate dehydrogenase reaction. Reaction of an enzyme snlfliydryl with the carbonyl carbon of glyceraldehyde-3-P forms a thiohemiacetal, which loses a hydride to NAD to become a thloester. Phosphorolysls of this thloester releases 1,3-blsphosphoglycerate. 

Baker, M.S., Feigan, J. and Lowther, D.A. (1989). The mechanisms of chondrocyte hydrogen peroxide damage. Depletion of intracellular ATP due to suppression of glycolysis caused by oxidation of glyceraldehyde-3-phosphate dehydrogenase. J. Rheumatol. 16, 7-14. 

Parker D.J., and Allison, W.S. (1969) The mechanism of inactivation of glyceraldehyde 3-phosphate dehydrogenase by tetrathionate, o-iodosobenzoate, and iodine monochloride. J. Biol. Chem. 244, 180-189. 

Figure 5.11 Mechanism of the glyceraldehyde-3 -phosphate dehydrogenase reaction. (From Voet and Voet, 2004. Reproduced with permission from John Wiley Sons., Inc.)...

Velick, S.F. (1954). The alcohol and glyceraldehyde-3-phosphate dehydrogenases of yeast and mammals. In The Mechanism of Enzyme Action. (McElroy, W.D. Glass, B., Eds.), pp. 491-519. The Johns Hopkins Press, Baltimore. 

A three-substrate (A, B, and C), two-product (P and Q) enzyme reaction scheme in which all substrates and products bind and are released in an ordered fashion. Glyceraldehyde-3-phosphate dehydrogenase has been reported to have this reaction scheme. The steady-state and rapid equilibrium expressions, in the absence of products and abortive complexes, are identical to the ordered Ter Ter mechanism. See Ordered Ter Ter Mechanism... 

Revises the presentation of the mechanism of glyceraldehyde 3-phosphate dehydrogenase. 

The most confusing aspect of the pathway proposed by Ochoa and his group now rests with the NAD requirement. In proceeding from L-malic acid to L-lactic acid, there is no net change in oxidation state. Yet in whole cells or cell-free extracts, the malo-lactic fermentation will not proceed in the absence of NAD. Therefore, by the proposed mechanism, one is unable to demonstrate the appearance of reduced cofactor, and the NAD specificity cannot be explained as a redox requirement. However, in the time since this mechanism was proposed, an NAD dependent enzyme (glyceraldehyde-3-phosphate dehydrogenase) has been described which requires NAD in a non-redox capacity (29), and it is possible that the same is true for the enzyme causing the malic acid-lactic acid transformation. 

A requirement for all fermentations is the existence of a mechanism for coupling ATP synthesis to the fermentation reactions. In the lactic acid and ethanol fermentations this coupling mechanism consists of the formation of the intermediate 1,3-bisphosphoglycerate by the glyceraldehyde 3-phosphate dehydrogenase (Fig. 10-3, step a). This intermediate contains parts of both the products ATP and lactate or ethanol. 

Irreversible inhibitors often provide clues to the nature of the active site. Enzymes that are inhibited by iodo-acetamide, for example, frequently have a cysteine in the active site, and the cysteinyl sulfhydryl group often plays an essential role in the catalytic mechanism (fig. 7.18). An example is glyceraldehyde 3-phosphate dehydrogenase, in which the catalytic mechanism begins with a reaction of the cysteine with the aldehyde substrate (see fig. 12.21). As we discuss in chapter 8, trypsin and many related proteolytic enzymes are inhibited irreversibly by diisopropyl-fluorophosphate (fig. 7.18), which reacts with a critical serine residue in the active site. 

Answer Pyruvate dehydrogenase is located in the mitochondrion, and glyceraldehyde 3-phosphate dehydrogenase in the cytosol. Because the mitochondrial and cytosolic pools of NAD are separated by the inner mitochondrial membrane, the enzymes do not compete for the same NAD pool. However, reducing equivalents are transferred from one nicotinamide coenzyme pool to the other via shuttle mechanisms (see Problem 21). 

Answer Ribulose 5-phosphate kinase, fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and glyceraldehyde 3-phosphate dehydrogenase would be inhibited. All have mechanisms requiring activation by reduction of a critical disulfide bond to a pair of —SH groups. Iodoacetate reacts irreversibly with free —SH groups. 

M22. Mohr, S., Stamler, J. S., and Brune, B., Mechanism of covalent modification of glyceraldehyde-3-phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents. FEBS Lett. 348, 223-227 (1994). 

Yang J, Gibson B, Snider J, Jenkins CM, Han X, Gross RW. Sub- 25. micromolar concentrations of palmitoyl-coa specifically thioes-terify cysteine 244 in glyceraldehyde-3-phosphate dehydrogenase inhibiting enzyme activity a novel mechanism potentially 26. underlying fatty acid induced insulin resistance. Biochemistry 2005 44 11903-11912. 

Let us consider the mechanism of glyceraldehyde 3-phosphate dehydrogenase in detail (Figure 16.8). In step 1, the aldehyde substrate reacts with the sulfhydryl group of cysteine 149 on the enzyme to form a hemithioacetal. Step 2 is the transfer of a hydride ion to a molecule of NAD + that is tightly bound to the enzyme and is adjacent to the cysteine residue. This reaction is favored by the deprotonation of the hemithioacetal by histidine 176. The products of this reaction are the reduced coenzyme NADH and a thioester intermediate. This thioester intermediate has a free energy close to that of the reactants. In step 3, orthophosphate attacks the thioester to form 1,3-BPG and free the cysteine residue. This displacement occurs only after the NADH formed from the aldehyde oxidation has left the enzyme and been replaced by a second NAD+. The positive charge on the NAD+ may help polarize the thioester intermediate to facilitate the attack by orthophosphate. 

Figure 16.8. Catalytic Mechanism of Glyceraldehyde 3-Phosphate Dehydrogenase. The reaction proceeds through a...

C. Lind, R. Gerdes, I. Schuppe-Koistinen, and LA. Cotgreave, Studies on the mechanism of oxidative modification of human glyceraldehyde-3-phosphate dehydrogenase by glutathione catalysis by glutaredoxin, Biochem. Biophys. Res. Commun. 247 (1998) 481-486. 

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