Reactivity of the Sulfhydryl Groups of Soluble Succinate Dehydrogenase
- 1 April 1976
- journal article
- Published by Wiley in European Journal of Biochemistry
- Vol. 63 (2), 365-371
- https://doi.org/10.1111/j.1432-1033.1976.tb10238.x
Abstract
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 by alkylating reagent in the presence of succinate or malonate suggests that N-ethylmaleimide acts as a sitedirected 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 °C in 50 mM Tris sulfate 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.Keywords
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