Reactivity of the sulfhydryl groups of soluble succinate dehydrogenase.
Soluble succinate dehydrogenase prepared by butanol extraction reacts with N-ethylmaleimide according to first-order kinetics with respect to both remaining active enzyme and the inhibitor concentration. Binding of the sulfhydryl groups of the enzyme prevents its alkylation by N-ethylmaleimide and inhibition by oxaloacetate. A kinetic analysis of the inactivation of alkylating reagent in the presence of succinate or malonate suggests that N-ethylmaleimide acts as a site-directed inhibitor. The apparent first-order rate constant of alkylation increases between pH 5.8 and 7.8 indicating a pKa value for the enzyme sulfhydryl group equal to 7.0 at 22 degrees C in 50 mM Tris-sufate buffer. Certain anions (phosphate, citrate, maleate and acetate) decrease the reactivity of the enzyme towards the alkylating reagent. Succinate/phenazine methosulfate reductase activity measured in the presence of a saturating concentration of succinate shows the same pH-dependence as the alkylation rate by N-ethylmaleimide. The mechanism of the first step of succinate oxidation, including a nucleophilic attack of substrate by the active-site sulfhydryl group, is discussed.
D001665:Binding Sites; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008314:Malonates / Q000494:pharmacology; D008433:Mathematics; D011485:Protein Binding; D012995:Solubility; D013385:Succinate Dehydrogenase / Q000378:metabolism; D013386:Succinates / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000032:analysis
D008314:Malonates; D013386:Succinates; D013438:Sulfhydryl Compounds; D013385:Succinate Dehydrogenase; D005033:Ethylmaleimide