Izbicka-Dimitrijević E, Gilbert H F
Biochemistry. 1984 Sep 11;23(19):4318-24. doi: 10.1021/bi00314a010.
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.
在pH 7.5的0.2 M磷酸盐缓冲液中，用二硫键试剂5,5'-二硫代双（2-硝基苯甲酸）（DTNB）、2,2-二硫代吡啶和4,4-二硫代吡啶使猪心硫解酶I失活，反应遵循二级动力学，速率常数分别为2.2×10²、25×10²和5.8×10² M⁻¹ min⁻¹。在pH 7.5时，化学计量浓度的硫醇氧化试剂偶氮二羧酸二乙酯在不到1分钟内就能使硫解酶失活。底物乙酰乙酰辅酶A的饱和浓度的存在或乙酰化酶（一种正常的催化中间体）的形成能显著保护硫解酶不被DTNB、2,2-二硫代吡啶和偶氮二羧酸二乙酯失活。天然硫解酶的所有五个巯基残基都能与任何一种二吡啶二硫化物反应，但即使在高浓度和长时间孵育的情况下，与DTNB反应的残基数量也仅相当于3.2个。硫解酶与DTNB的反应会导致形成1.0 - 1.4摩尔的链内二硫键和0.65摩尔的混合二硫键。用等摩尔浓度的偶氮二羧酸二乙酯使硫解酶失活后，每个亚基会形成1.2摩尔的链内二硫键。硫解酶与DTNB或偶氮二羧酸二乙酯反应不会导致亚基之间发生交联。DTNB失活的酶可用过量的二硫苏糖醇重新激活，而偶氮二羧酸二乙酯失活的酶完全抵抗二硫苏糖醇的重新激活。根据所使用的氧化剂不同，似乎至少有两种不同的方式形成无活性的氧化酶产物，这表明活性位点处或其附近可能存在多个巯基。
