Glyceraldehyde-3-phosphate dehydrogenase inhibition

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. 

Molina, Y., Vedia, L., McDonald, B., Keep, B., Briine, B., DiSilvio, M., Billiar, T. R., and Lapetina, E. G. (1993). Nitric oxide-induced 5-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase inhibits enzymatic activity and increases endogenous ADP-ribosylation. J. Biol. Chetn. 267, 24929-24932. 

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). 

Brune and Lapetina (1989) reported that NO could activate a platelet ADP-ribosyltransferase that resulted in the ribosylation of a 39 kDa protein. Subsequent work revealed that the protein was glyceraldehyde phosphate dehydrogenase (GAP-DH), and that ribosylation was associated with reduced GAP-DH activity (Dimmeler et al., 1992). In our collaboration with Molina et al., (1992), we have shown that GAP-DH activity is dramatically inhibited in C. parvum treated rats and that this action is associated with both a ribosylation and nitro-sylation of the enzyme. Such a marked inhibition of a glycolytic enzyme could explain some of the metabolic changes observed in the liver in sepsis. 

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. 

The basis of the action of iodoacetate on muscle contraction was uncovered by Dickens and Rapkine (ca. 1933). They found iodoacetate alkylated SH groups on proteins, especially those in glyceraldehyde 3-phosphate dehydrogenase (G-3-PDH). When the enzyme was inhibited precursors accumulated—hexose mono- and diphosphates—as in Lundsgaard s experiments. 

Glyceraldehyde-3- phosphate dehydrogenase (bacteria) HA inhibits (Ki = 4.7 mM) the oxidation of glyceraldehyde-3-phosphate to 3-phosphoglycerate 54... 

Dimmeler, S., Lottspeich, F., and Briine, B. (1992). Nitric oxide causes ADP-ribosylation and inhibition of glyceraldehyde-3-phosphate dehydrogenase. J. Biol. Chem. 267, 16771-16774. 

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. 

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. 

Zinc also inhibits a variety of nonmetalloenzymes. Inhibition constants in the nM range are observed in some cases. In these situations it is possible that the zinc could be involved in regulating the activity of the enzyme. Thionein has been demonstrated to reverse the inhibition glyceraldehyde-3-phosphate dehydrogenase by zinc ions, suggesting the possibility that thionein (apo-metaUothionien see Metallothioneins) can be involved in a regulatory capacity with zinc as its partner. ... 

More recently, there has been direct observation of protein structural perturbation in the frozen state using phosphorescence lifetime measurements [62]. Reductions in this parameter indicated that freezing perturbed the tertiary structure (at a protein concentration of 3-5 pM) of azurin, ribonuclease, alcohol dehydrogenase, alkaline phosphatase, glyceraldehyde 3-phosphate dehydrogenase, and LDH. The cryoprotectants sucrose and glycerol were tested and were found to inhibit the freezing-induced structural perturbations, with almost complete protection noted at a 1 M concentration. 

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