The reaction of N-ethylmaleimide at the active site of succinate dehydrogenase.
Since 1938 mammalian succinate dehydrogenase has been thought to contain thiol groups at the active site. This hypothesis was questioned recently, because irreversible inhibition by bromopyruvate and N-ethylmaleimide appeared not to satisfy the requisite criteria for reaction at the active site. These recent observations of incomplete inactivation of succinate dehydrogenase by N-ethylmaleimide and incomplete protection by substrates can, however, be explained adequately by the presence of oxalacetate and other strong competitors of the inactivation process in the enzyme used in these studies. Substrates, competitive inhibitors, and anions which activate succinate dehydrogenase protect the enzyme from inhibition by N-ethylmaleimide. Inhibition of succinate dehydrogenase by N-ethylmaleimide involves at least two second order reactions which are pH dependent, with pKa values of 8.0 to 8.2. This pH dependence, the known reactivity of N-ethylmaleimide toward thiols, and the protection by substrate and competitive inhibitors indicate that sulfhydryl residues are required for catalytic activity and perform an essential, not secondary, role in the catalysis. Just as the presence of tightly bound oxalacetate prevents inhibition by N-ethylmaleimide, alkylation of the sulfhydryl residue(s) at the active site prevents the binding of [14C]oxalacetate. Thus, these thiol groups at the active site also may be the site of tight binding of oxalacetate during the activation-deactivation cycle.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005033:Ethylmaleimide / Q000494:pharmacology; D005650:Fumarates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008293:Malates / Q000494:pharmacology; D008314:Malonates / Q000494:pharmacology; D008433:Mathematics; D008931:Mitochondria, Muscle / Q000201:enzymology; D009206:Myocardium; D010071:Oxaloacetates / Q000494:pharmacology; D011485:Protein Binding; D013385:Succinate Dehydrogenase / Q000378:metabolism; D013386:Succinates / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D005650:Fumarates; D008293:Malates; D008314:Malonates; D010071:Oxaloacetates; D013386:Succinates; D013385:Succinate Dehydrogenase; D005033:Ethylmaleimide