Multiple oxidation products of sulfhydryl groups near the active site of thiolase I from porcine heart.
The inactivation of porcine heart thiolase I with the disulfide reagents 5,5'-dithiobis(2-nitrobenzoate) (DTNB) and 2,2- and 4,4-dithiopyridine in 0.2 M phosphate buffer, pH 7.5, follows second-order kinetics with rate constants of 2.2 X 10(2), 25 X 10(2), and 5.8 X 10(2) M-1 min-1, respectively. Stoichiometric concentrations of the thiol-oxidizing reagent diethyl azodicarboxylate inactivate thiolase in less than 1 min at pH 7.5. The presence of saturating concentrations of the substrate acetoacetyl coenzyme A or the formation of the acetyl enzyme (a normal catalytic intermediate) results in a significant protection against the inactivation of thiolase by DTNB, 2,2-dithiopyridine, and diethyl azodicarboxylate. All five sulfhydryl residues of native thiolase react with either of the dipyridyl disulfides, but only the equivalent of 3.2 residues react with DTNB even at high concentrations and prolonged incubation times. The reaction of thiolase with DTNB leads to the formation of 1.0-1.4 mol of intrachain disulfide and 0.65 mol of mixed disulfides. After inactivation of thiolase with an equimolar concentration of diethyl azodicarboxylate, 1.2 mol of intrachain disulfide per subunit is found. No cross-linking between the subunits occurs as a result of the reaction of thiolase with DTNB or diethyl azodicarboxylate. The DTNB-inactivated enzyme can be reactivated with excess dithiothreitol while the diethyl azodicarboxylate inactivated enzyme is totally resistant to reactivation by dithiothreitol. There appear to be at least two different ways of forming inactive, oxidized enzyme products depending on the oxidant used, suggesting the possibility of multiple sulfhydryl groups at or near the active site.
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000494:pharmacology; D000101:Acetyl-CoA C-Acetyltransferase / Q000378:metabolism*; D000123:Acetyltransferases / Q000378:metabolism*; D000818:Animals; D001391:Azo Compounds / Q000494:pharmacology; D001665:Binding Sites; D004220:Disulfides; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology*; D011725:Pyridines / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013552:Swine
D001391:Azo Compounds; D004220:Disulfides; D011725:Pyridines; D013438:Sulfhydryl Compounds; C043515:diazocarboxylic acid ethyl ester; C001105:2,2'-dipyridyl disulfide; C007542:4,4'-dipyridyl disulfide; D015082:2,2'-Dipyridyl; D004228:Dithionitrobenzoic Acid; D000123:Acetyltransferases; D000101:Acetyl-CoA C-Acetyltransferase