Glyceraldehyde-3-phosphate dehydrogenase active site

The inhibitory effects of heavy metals, and of cyanide on cytochrome oxidase and of arsenate on glyceraldehyde phosphate dehydrogenase, are examples of non-competitive inhibition. This type of inhibitor acts by combining with the enzyme in such a way that for some reason the active site is rendered inoperative. The inhibition may or may not be reversible but it is not affected by the addition of extra substrate. 

Thus, an initial drop in ATP is followed by increases in Ca2+, which inhibits ATP synthase and increases ROS and reactive nitrogen species (RNS) formation via xanthine oxidase. These inhibit thiol-dependent Ca2+ transport. The reactive molecules can also inhibit the electron transport chain (by reacting with Fe at the active sites) and enzymes in glycolysis, notably glyceraldehyde 3-phosphate dehydrogenase, leading to further losses of ATP. The depleted ATP exacerbates the intracellular Ca2 increase as a result of reduced transport out and sequestration into the endoplasmic reticulum. 

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. 

Glyceraldehyde-3-phosphate dehydrogenase has an essential cysteine residue in its active site. The enzyme forms a transient acyl compound with its substrate, glyceraldehyde 3-phosphate, (a) What is the general chemical name of the compound (b) Draw its likely structure. 

Fig. 19. The active sites of lactate dehydrogenase (A) and glyceraldehyde-3-phosphate dehydrogenase (B). From the work of Rossmann and colleagues [52],...

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). 

Several different amino acid side chains can act as nucleophiles in enzyme catalysis. The most powerful nucleophile is the thiol side chain of cysteine, which can be deproto-nated to form the even more nucleophilic thiolate anion. One example in which cysteine is used as a nucleophile is the enzyme glyceraldehyde 3-phosphate dehydrogenase, which uses the redox coenzyme NAD+. As shown in Fig. 10, the aldehyde substrate is attacked by an active site cysteine, Cys-149, to form a hemi-thioketal intermediate, which transfers hydride to NAD+ to form an oxidized thioester intermediate (7). Attack of phosphate anion generates an energy-rich intermediate 3-phosphoglycerate. 

Figure 16.7. Structure of Glyceraldehyde 3-Phosphate Dehydrogenase. The active site includes a cysteine residue and a histidine residue adjacent to a hound NAD+. 

Enzymes that contain free sulfhydryl groups at the active site (e.g., glyceraldehyde-3-phosphate dehydrogenase see Chapter 13) react with an alkylating reagent, iodoacetic acid, resulting in inactivation of the enzyme. 

Dietze, E. C., Schafer, A., Omichinski, J. G., Nelson, S. D. Inactivation of glyceraldehyde-3-phosphate dehydrogenase by a reactive metabolite of acetaminophen and mass spectral characterization of an arylated active site peptide. Chertr Res. Toxicol. 1997,10,1097-1103. 

Figure 16.6 Structure of glyceraldehyde 3-phosphate dehydrogenase. Notice that the active site includes a cysteine residue and a histidine residue adjacent to a bound NAD molecule. The sulfur atom of cysteine will link with the substrate to form a transitory thioester intermediate. [Drawn from IGAD.pdb,]...

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