Nicotinamide Adenine Dinucleotide As an Active Site Director in Glyceraldehyde 3-Phosphate Dehydrogenase Modification

Abstract

Abstract Multivalent anions, sulfate, phosphate, or arsenate, selectively decrease the rate of alkylation of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase by both iodoacetamide and iodoacetate. N-Acetylation of the lysine residue that in the apoenzyme neighbors the essential cysteine residue does not eliminate this anion effect, suggesting that an alternative unique enzyme site is responsible for binding these anions. NAD+ (or 3-acetylpyridine adenine dinucleotide) at high concentration strikingly reverses this effect with iodoacetate; with iodoacetamide a further retardation in alkylation rate is observed. It is proposed that binding of NAD+ limits access of iodoacetamide or other uncharged reagents to the active site sulfhydryl group and simultaneously produces, via the pyridinium cation, a site capable of binding iodoacetate or any other negatively charged reagent. The role of NAD+ in acyl phosphatase and p-nitrophenyl hydrolase activities is considered in this context. The dependence on coenzyme concentration of the alkylation reaction for both reagents was determined with apoenzyme in various media. The results at low NAD+ concentrations in the presence of a multivalent anion indicate that a given coenzyme molecule facilitates, by means of the specific coulombic interaction, carboxymethylation (and not carbamylmethylation) at each of the sites to which it is bound during the reaction period. Finally the possible use of NAD+ (NADP+) as an active site director is commented upon in terms of modifying enzymatic behavior in vivo.