Glyceraldehyde 3-phosphate dehydrogenase thiol group

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). 

Bloxham, D.P., and Sharma, R.P. (1979) The development of 5,5 -polymethylenebis(methanethiosulfona tes) as reversible cross-linking reagent for thiol groups and their use to form stable catalytically active cross-linked dimers with glyceraldehyde-3-phosphate dehydrogenase. Biochem. J. 181, 355. 

Addition reactions often occur as parts of more complex reactions. For example, a thiol group of glyceraldehyde-3-phosphate dehydrogenase reacts with the aldehyde substrate to form a hemimercaptal, which is subsequently oxidized to a thioester (see Fig. 15-6). 

In enzymes, the most common nucleophilic groups that are functional in catalysis are the serine hydroxyl—which occurs in the serine proteases, cholinesterases, esterases, lipases, and alkaline phosphatases—and the cysteine thiol—which occurs in the thiol proteases (papain, ficin, and bromelain), in glyceraldehyde 3-phosphate dehydrogenase, etc. The imidazole of histidine usually functions as an acid-base catalyst and enhances the nucleophilicity of hydroxyl and thiol groups, but it sometimes acts as a nucleophile with the phos-phoryl group in phosphate transfer (Table 2.5). 

Nicotinamide-(S-methylmercury-thioinosine) dinucleotide was found to exhibit coenzyme properties with lactate dehydrogenase and liver alcohol dehydrogenase, but inactivate yeast alcohol dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase an essential thiol group was therefore modified in the last two cases. 

The administration of TNT to laboratory animals leads to the excretion of 4-NHOH-DNT, 2-NH2-DNT, and 4-NH2-DNT in the urine [59], and to the formation of covalent adducts with microsomal liver and kidney proteins, hemoglobin, and other blood proteins [60], The acid hydrolysis of adducts yielded mainly 2-NH2-DNT (2-ADNT) and 4-NH2-DNT (4-ADNT). Incubation of rat liver microsomes with TNT and NADPH under aerobic conditions resulted in the formation of NH2-DNTs and the transient metabolite 4-NHOH-DNT [57], The formation of covalent protein adducts with TNT metabolites was enhanced by the presence of 02 and decreased by GSH. This is consistent with the scheme of the TNT adduct formation with the central role of the nitroso metabolite (NO-DNT) reaction with protein or nonprotein thiols (RSH Equation 9.11) [57], The acid hydrolysis of the sulfinamide adduct (RS(0)-NH-DNT) formed after the rearrangement of the semimercaptal (RS-N(OH)-DNT Equation 9.12) will yield NH2-DNT. The mixture of NHOH-DNTs inhibits bacterial glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase more efficiently than TNT [61]. This was attributed to the covalent modification of protein -SH groups. 

A cartoon representation of the oxidation path used in Equation 9.5 is shown in Scheme 9.5. The process is catalyzed by the enzyme glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). A large body of evidence has been accumulated about the role of the enzyme and, in particular, that the catalytic site on the enzyme that is used to help the oxidation occur bears a thiol (-SH, Chapter 8) group (from the amino acid cysteine. Chapter 12). Further, it has been shown that hydride (H ) transfer to nicotinamide dinucleotide (NAD ") (see Chapter 12), a required cofactor that accounts for the transfer of the hydride anion and, thus, the oxidation itself, is also involved. 

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