Inhibition of D(--)-beta-hydroxybutyrate dehydrogenase by modifiers of disulfides, thiols, and vicinal dithiols.
D(--)-beta-hydroxybutyrate dehydrogenase of beef heart mitochondria catalyzes the reversible oxidation of D-(--)-beta-hydroxybutyrate to acetoacetate in the presence of NAD. Both the membrane-bound and the soluble forms of the enzyme are inhibited by modifiers of thiols [N-ethylmaleimide (NEM) and p-(chloromercuri)phenylsulfonate (pCMS)], vicinal dithiols [phenylarsine oxide and diazenedicarboxylic acid bis(dimethylamide) (diamide)], and disulfides (sulfite, sulfide, and cyanide). NAD and NADH, but not beta-hydroxybutyrate and acetoacetate, protect the enzyme against inhibition by NEM, pCMS, phenylarsine oxide, and diamide. As tested with NEM and diamide, the inhibitions caused by mono- and dithiol modifiers were pseudo first order, and the reaction order with respect to the concentration of either inhibitor was unity, thus indicating the modification of a single essential thiol and/or dithiol. Sulfite and sulfide inhibitions appeared to be competitive with respect to beta-hydroxybutyrate, with Ki values of 10-15 and about 240 microM, respectively. Sulfite inhibition was uncompetitive with respect to NAD, NADH, and acetoacetate. The above results have suggested the presence in D(--)-beta-hydroxybutyrate dehydrogenase of an essential thiol and/or a vicinal dithiol associated with the binding site(s) of NAD and NADH. The inhibition by sulfite, sulfide, and cyanide might be indicative of the presence of an essential disulfide or due to a ternary complex formation involving the enzyme, NAD, and the above nucleophiles.
D000090:Acetoacetates; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology*; D001391:Azo Compounds / Q000494:pharmacology*; D002417:Cattle; D003958:Diamide / Q000494:pharmacology*; D004220:Disulfides; D006884:Hydroxybutyrate Dehydrogenase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D013367:Submitochondrial Particles / Q000201:enzymology; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013440:Sulfides / Q000494:pharmacology*; D013447:Sulfites / Q000494:pharmacology*
D000090:Acetoacetates; D001152:Arsenicals; D001391:Azo Compounds; D004220:Disulfides; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D013440:Sulfides; D013447:Sulfites; C029341:oxophenylarsine; D003958:Diamide; D006884:Hydroxybutyrate Dehydrogenase