Alkylation of cysteinyl residues of pig heart NAD-specific isocitrate dehydrogenase by iodoacetate.
Pig heart NAD-specific isocitrate dehydrogenase is inactivated by reaction with iodoacetate at pH 6.0. Loss of activity can be attributed to the formation of 1-2 mol of carboxymethyl-cysteine per peptide chain. The rate of inactivation is markedly decreased by the combined addition of Mn2+ and isocitrate, but not by alpha-ketoglutarate, the coenzyme NAD or the allosteric activator ADP. The substrate concentration dependence of the decreased rate of inactivation yields a dissociation constant of 1.6 mM for the enzyme-manganous-dibasic isocitrate complex, a value that is 50 times higher than the Km for this substrate. This result suggests that in protecting the enzyme against iodoacetate, isocitrate may bind to a region distinct from the catalytic site. Isocitrate and Mn2+ also prevent thermal denaturation, with an affinity for the enzyme close to that observed for the iodoacetate-sensitive site. The alkylatable cysteine residues may contribute to a manganous-isocitrate binding site which is responsible for stabilizing an active conformation of the enzyme.
D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000032:analysis; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000494:pharmacology; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology*; D009243:NAD; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013552:Swine
D007461:Iodoacetates; D009243:NAD; D008345:Manganese; D007521:Isocitrate Dehydrogenase; D003545:Cysteine