Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with 3-bromopropionic acid.
Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with 3-bromopropionic acid.
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通过用 3-溴丙酸修饰半胱氨酸残基 174 灭活马肝乙醇脱氢酶。
DOI：
10.1021/bi00297a007
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发表时间：
1984
期刊：
影响因子：
2.9
通讯作者：
B. Plapp
中科院分区：
文献类型：
--
作者：
V. K. Chadha;B. Plapp
马肝酒精脱氢酶在pH 7和25℃条件下被3-溴丙酸用米切里斯动力学灭活。无NAD+时，Ki为2 mM，伪双分子速率常数（k3/Ki）为0.03 M-1 s-1；在1 mM NAD+存在下，Ki为2.3 mM， k3/Ki为0.006 M-1 s-1。3-溴丙酸是一种竞争性抑制剂，Ki为0.4 mM，对抗乙醇作为底物。与NAD+ X吡唑和NADH X异丁酰胺的三元配合物的失活被阻止，被NAD+， NADH或联吡啶延缓，并且几乎不受咪唑和AMP的影响。羧乙基化酶不能检测到（通过分光光度法观察到）结合联吡啶，NAD+或NADH。用放射性的3-溴丙酸灭活酶，氨基乙基化，用胰蛋白酶和凝乳胰蛋白酶消化。对标记肽的分析表明，Cys-174主要被修饰。在1mm NAD+的存在下，反应的特异性大大降低。3-溴丙酸的羧基与Arg-369的胍基的相互作用可能促进了与Cys-174的选择性反应，Cys-174在活性位点与锌结合。羧乙基化通过干扰辅酶的焦磷酸盐与酶的适当结合而明显失活。
Horse liver alcohol dehydrogenase is inactivated with Michaelis kinetics at pH 7 and 25 degrees C by 3-bromopropionic acid. In the absence of NAD+, the Ki is 2 mM, and the pseudo bimolecular rate constant (k3/Ki) is 0.03 M-1 s-1; in the presence of 1 mM NAD+, Ki is 2.3 mM, and k3/Ki is 0.006 M-1 s-1. 3-Bromopropionic acid is a competitive inhibitor, Ki of 0.4 mM, against ethanol as a substrate. Inactivation was prevented in the ternary complexes with NAD+ X pyrazole and NADH X isobutyramide, was retarded by NAD+, NADH, or bipyridine, and was almost unaffected by imidazole and AMP. Carboxyethylated enzyme did not detectably (as observed spectrophotometrically) bind bipyridine, NAD+, or NADH. Enzyme was inactivated with radioactive 3-bromopropionic acid, aminoethylated, and digested with trypsin and chymotrypsin. Analysis of the labeled peptides showed that Cys-174 was predominantly modified. In the presence of 1 mM NAD+, the reaction was much less specific. The interaction of the carboxyl group of 3-bromopropionic acid with the guanidino group of Arg-369 probably facilitates the selective reaction with Cys-174, which is ligated to the zinc at the active site. Carboxyethylation apparently inactivates by interfering with the proper binding of the pyrophosphate of the coenzyme to the enzyme.