Jones D T, Khalifah R G
Adv Exp Med Biol. 1980;132:77-83. doi: 10.1007/978-1-4757-1419-7_9.
Horse liver alcohol dehydrogenase was carboxymethylated at the active site Cys-46 using 90% [1-13C]bromoacetate. The enriched carboxylate resonance was studied in the absence and presence of coenzymes and inhibitors. Previous ambiguities regarding the resonance in the NAD+ complex have now been resolved. However, it is shown that imidazole, an inhibitor introduced during the carboxymethylation, is not removed by the standard gel filtration step frequently employed and must thus bind much more tightly to the enzyme than suspected. Competition experiments involving imidazole and halide inhibitors show that the imidazole is preferentially bound when both are present in equimolar amounts. This suggests that the crystallographically identified anion binding site at the zinc of the carboxymethylated enzyme may require re-evaluation. The electron density at the zinc of the modified enzyme may be better explained as being due to unsuspected binding of imidazole.
使用90%的[1-13C]溴乙酸盐对马肝乙醇脱氢酶活性位点的半胱氨酸-46进行羧甲基化修饰。在有无辅酶和抑制剂的情况下，对富集的羧酸盐共振进行了研究。先前关于NAD+复合物中共振的模糊问题现已得到解决。然而，研究表明，在羧甲基化过程中引入的抑制剂咪唑，不能通过常用的标准凝胶过滤步骤去除，因此它与酶的结合必定比预期的更紧密。涉及咪唑和卤化物抑制剂的竞争实验表明，当两者等摩尔存在时，咪唑优先结合。这表明，经羧甲基化修饰的酶中锌离子处晶体学鉴定的阴离子结合位点可能需要重新评估。修饰后酶中锌离子处的电子密度，或许可以更好地解释为是由于咪唑意外的结合所致。
