Klinman J P
Biochemistry. 1975 Jun 17;14(12):2568-74. doi: 10.1021/bi00683a002.
Yeast alcohol dehydrogenase is inactivated and alkylated by styrene oxide in a single exponential kinetic process. The concentration dependence of half-times for inactivation indicates the formation of an enzyme inhibitor complex, KI = 2.5 times 10(-2) M at pH 8.0. Reduced nicotinamide adenine dinucleotide (NADH), at a concentration of 3 times 10(-4) M where Kd congruent to 1 times 10(-5) M, has a small effect on kinetic parameters for inactivation. Although benzyl alcohol and acetamide-NADH increase the KI for styrene oxide in a manner consistent with their dissociation constants, substrate also increases the rate of inactivation at high styrene oxide concentrations. The reciprocal of half-times for inactivation, extrapolated to infinite styrene oxide concentration, increases with pH between 7.6 and 9.0, pK congruent to 8.5. The stoichiometry of alkylation by [3H]styrene oxide is 2.2 mol of reagent incorporated/mol of subunit, and is accompanied by the loss of 1.9 mol of sulfhydryl/mol of subunit; prior alkylation with iodoacetamide reduces the stoichiometry to 0.88:1, and increases the rate of labeling. Tryptic digests of enzyme modified with [14C]iodoacetamide or [3H]styrene oxide produce two major peptides which cochromatograph, indicating that styrene oxide and iodoacetamide modify the same cysteine residues. Previous investigators have reported that iodoacetate, iodoacetamide, and butyl isocyanate alkylate either of two reactive cysteines of yeast alcohol dehydrogenase; both cysteines cannot be modified simultaneously [Belke et al. (1974), Biochemistry 13, 3418]. The inactivation of enzyme by p-chloromercuribenzoate (PCMB) is reported here to be accompanied by the incorporation of 2.3 mol of PCMB/mol of enzyme subunits, in analogy with styrene oxide; the planarity of the alkylating agent appears to be an important factor in determining the stoichiometry of labeling.
酵母乙醇脱氢酶在单一指数动力学过程中被环氧苯乙烯失活并烷基化。失活半衰期的浓度依赖性表明形成了一种酶抑制剂复合物，在pH 8.0时，解离常数KI = 2.5×10⁻² M。浓度为3×10⁻⁴ M（解离常数Kd约为1×10⁻⁵ M）的还原型烟酰胺腺嘌呤二核苷酸（NADH）对失活动力学参数的影响较小。虽然苯甲醇和乙酰酰胺 - NADH以与其解离常数一致的方式增加了环氧苯乙烯的KI，但在高环氧苯乙烯浓度下，底物也会增加失活速率。失活半衰期的倒数外推至无限环氧苯乙烯浓度时，在pH 7.6至9.0之间随pH升高而增加，pK约为8.5。[³H]环氧苯乙烯的烷基化化学计量比为每摩尔亚基掺入2.2摩尔试剂，同时每摩尔亚基损失1.9摩尔巯基；用碘乙酰胺预先烷基化会使化学计量比降至0.88:1，并增加标记速率。用[¹⁴C]碘乙酰胺或[³H]环氧苯乙烯修饰的酶的胰蛋白酶消化产物产生两个共色谱的主要肽段，表明环氧苯乙烯和碘乙酰胺修饰相同的半胱氨酸残基。先前的研究人员报道，碘乙酸、碘乙酰胺和异氰酸丁酯会烷基化酵母乙醇脱氢酶的两个反应性半胱氨酸中的任意一个；两个半胱氨酸不能同时被修饰[Belke等人（1974年），《生物化学》13, 3418]。本文报道对氯汞苯甲酸（PCMB）使酶失活时，每摩尔酶亚基掺入2.3摩尔PCMB，这与环氧苯乙烯类似；烷基化剂的平面性似乎是决定标记化学计量比的一个重要因素。
