Evidence for the existence of two classes of sulfhydryl groups essential for membrane-bound succinate dehydrogenase activity.
Kinetics of the inhibition of activated membrane-bound dehydrogenase by N-substituted maleimides were studied. Three maleimide derivatives having a different hydrophobic character (N-ethyl-, N-butyl-, and N-benzylmaleimide) were tested. The method developed by Ray & Koshland (Ray, W. J., Jr., & Koshland, D. E., Jr. (1961) J. Biol, Chem. 236, 1973-1979) was used for analyzing experimental data. The results showed that two classes of sulfhydryl groups, with quite different reactivities, were essential for catalytic activity. The most reactive sulfhydryl groups were located in the substrate site as revealed by the fact that they were protected against alkylation in the presence of succinate or a competitive inhibitor, malonate. However, ligands of the catalytic site did not completely prevent inactivation of succinate dehydrogenase. Analysis of the kinetics of the inhibition observed in the presence of substrate indicated that the slow-reacting sulfhydryl groups did not belong to the active site. Rate constant values of the reaction of each set of sulfhydryl groups with the three maleimide derivatives showed that the most reactive thiols were probably located in a hydrophobic microenvironment since alkylation of this set of sulfhydryl groups was sensitive to the hydrophobic character of the thiol reagent. The reactivity of the other class of sulfhydryl groups was not influenced by the nature of the substituent. When the enzyme was deactivated by oxaloacetate, the two classes of sulfhydryl groups became unreactive with the alkylating agents. Masking of these groups may reflect a conformational change of the enzyme.
D000818:Animals; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology*; D008928:Mitochondria / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D051381:Rats; D013329:Structure-Activity Relationship; D013367:Submitochondrial Particles / Q000201:enzymology*; D013385:Succinate Dehydrogenase / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000032:analysis*
D008301:Maleimides; D013438:Sulfhydryl Compounds; D013385:Succinate Dehydrogenase