Reactivity of the Sulfhydryl Groups of Soluble Succinate Dehydrogenase

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 Nethylmaleimide 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 sitcdirected inhibitor. The apparent first-order rate constant of alkylation increases between pH 5.8 and 7.8 indicating a pK, value for the enzyme sulfhydryl group equal to 7.0 at 22 "C in 50 mM Trissulfate 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.

Regulation of mitochondrial succinate dehydrogenase by substrate type activators

“…These conclusions are further supported by the difference between KmalonllL' ( 1 66 p M ) as calculated from the data presented in Figure 6B and K , for malonate ( 1 0 pM, Vinogradov et al, 1976;18 pM, Dervartanian and Veeger, 1964). Table I1 lists the K A~, values for the other ligands when reacting with the regulatory site.…”

“…The reaction of this SH group with MalNEt] is blocked by either activators or oxaloacetate (Kenney et al, 1976;Vinogradov et al, 1976). The dissociation constants of succinate and malonate, as calculated from their effect on rate of alkylation, are similar to the respective K , or K , values (Vinogradov et al, 1976).…”

“…Oxaloacetate and the enzyme form a very tight complex, which can be dissociated either by activation (Ackrell et al, 1974; or by denaturation. It was suggested that oxaloacetate is covalently linked as a thiohemiacetal (Vinogradov et al, 1972) with a SH group at the active site of the flavoprotein subunit of the enzyme (Kenney et al, 1976).The reaction of this SH group with MalNEt] is blocked by either activators or oxaloacetate (Kenney et al, 1976;Vinogradov et al, 1976). The dissociation constants of succinate and malonate, as calculated from their effect on rate of alkylation, are similar to the respective K , or K , values (Vinogradov et al, 1976).…”

“…The dissociation constants of succinate and malonate, as calculated from their effect on rate of alkylation, are similar to the respective K , or K , values (Vinogradov et al, 1976). Yet, these studies measured the effect of only one modulator, activator or oxaloacetate, on the reactivity of this SH group, but not their simultaneous interaction with the proposed common active site.…”

A succinate dehydrogenase with novel structure and properties from the hyperthermophilic archaeon Sulfolobus acidocaldarius: genetic and biophysical characterization

“…The conserved arginine at position 251 possibly provides a strong two-point interaction with the substrate carboxylates (53). The S. acidocaldarius flavoprotein lacks in this active-site region (residue 250) a putative reactive cysteine residue (59) which is conserved in sequences of B. taurus SdhA, E. coli SdhA and FrdA, and other flavoproteins. However, recent studies disproved that this cysteine residue is essential for catalysis (23,35,53,58).…”