Topham C M, Dalziel K
Eur J Biochem. 1986 Feb 17;155(1):87-94. doi: 10.1111/j.1432-1033.1986.tb09461.x.
Sheep liver 6-phosphogluconate dehydrogenase is shown to be inactivated by diethylpyrocarbonate in a biphasic manner at pH 6.0, 25 degrees C. After allowing for the hydrolysis of the reagent, rate constants of 56 M-1 s-1 and 11.0 M-1 s-1 were estimated for the two processes. The complete reactivation of partially inactivated enzyme by neutral hydroxylamine, the elimination of the possibility that modification of cysteine or tyrosine residues are responsible for inactivation, and the magnitudes of the rate constants for inactivation relative to the experimentally determined value for the reaction of diethylpyrocarbonate with N alpha-acetylhistidine (2.2 M-1 s-1), all suggested that enzyme inactivation occurs solely by modification of histidine residues. Comparison of the experimental plot of residual fractional activity versus the number of modified histidine residues per subunit with simulated plots for three hypothetical models, each predicting biphasic kinetics, indicated that inactivation results from the modification of at most one essential histidine residue per subunit, although it appears that other (non-essential) histidines react independently. This histidine is thought to be His-242 and is present in the active site. Evidence in support of its role in catalysis is briefly discussed. Both 6-phosphogluconate and organic phosphate protect against inactivation, and a kinetic analysis of the protection indicated a dissociation constant of 2.1 X 10(-6) M for the enzyme--6-phosphogluconate complex. NADP+ also protected, but this might be due, at least in part, to a reduction in the effective concentration of diethylpyrocarbonate.
已表明绵羊肝脏6-磷酸葡萄糖酸脱氢酶在pH 6.0、25℃下会被焦碳酸二乙酯以双相方式灭活。在考虑试剂水解后，估计两个过程的速率常数分别为56 M⁻¹ s⁻¹和11.0 M⁻¹ s⁻¹。用中性羟胺可使部分失活的酶完全复活，排除了半胱氨酸或酪氨酸残基修饰导致失活的可能性，且失活速率常数的大小相对于焦碳酸二乙酯与Nα-乙酰组氨酸反应的实验测定值（2.2 M⁻¹ s⁻¹），所有这些都表明酶失活仅通过组氨酸残基的修饰发生。将残余分数活性与每个亚基修饰的组氨酸残基数量的实验图与三个假设模型的模拟图进行比较，每个模型都预测双相动力学，结果表明失活是由于每个亚基最多修饰一个必需的组氨酸残基所致，尽管似乎其他（非必需）组氨酸独立反应。这个组氨酸被认为是His-242，存在于活性位点。简要讨论了支持其在催化中作用的证据。6-磷酸葡萄糖酸和有机磷酸盐都能防止失活，对这种保护作用的动力学分析表明酶 - 6-磷酸葡萄糖酸复合物的解离常数为2.1×10⁻⁶ M。NADP⁺也有保护作用，但这可能至少部分是由于焦碳酸二乙酯有效浓度的降低。
