Department of Biochemistry, The University of Iowa, Iowa City, IA, 52242, USA.
Chem Biol Interact. 2019 Apr 1;302:172-182. doi: 10.1016/j.cbi.2019.01.040. Epub 2019 Feb 2.
The catalytic zincs in complexes of horse liver and yeast alcohol dehydrogenases (ADH) with NAD and the substrate analogue, 2,2,2-trifluoroethanol, are ligated to two cysteine residues and one histidine residue from the protein and the oxygen from the alcohol. The zinc facilitates deprotonation of the alcohol and is essential for catalysis. In the yeast apoenzyme, the zinc is coordinated to a nearby glutamic acid, which is displaced by the alcohol in the complex with NAD. Some homologous medium chain dehydrogenases have a cysteine replaced by aspartic or glutamic acid residues. How an aspartic acid would affect catalysis was studied by replacing Cys-153 in Saccharomyces cerevisiae ADH1 by using site-directed mutagenesis. The C153D enzyme was about as stable as the wild-type enzyme, if EDTA was not included in the buffers. The substitution increased affinity for NAD by 3-fold, but did not affect NADH binding. At pH 7.3, the turnover number for ethanol oxidation (V/E) decreased by 7-fold and catalytic efficiency decreased 18-fold (V/EK), but turnover for acetaldehyde reduction (V/E) was the same as for wild-type enzyme and catalytic efficiency decreased 8-fold (V/EK). Deuterium isotope effects of 3.0 on V/E and 3.8 on V/EK for ethanol oxidation suggest that hydride transfer is more rate-limiting for turnover for the C153D enzyme than by wild-type enzyme. The patterns of pH dependence for V/EK for ethanol oxidation were similar for both enzymes in the pH range from 7 to 9. The C153D substitution decreased binding of trifluoroethanol by 5-fold and of pyrazole by 65-fold. Substrate specificities for C153D and wild-type ADHs for primary alcohols have similar patterns. Efficiency for secondary alcohols decreased only about 4-fold, and efficiencies for 1,2-propanediol and acetone were about the same as for wild-type enzyme. The C153D substitution modestly affects catalysis by altering ligand exchange on the zinc or local structure. Structures and mechanisms for acid-base catalysis in related medium chain dehydrogenases with substitutions of the homologous cysteine are reviewed and analyzed.
马肝和酵母醇脱氢酶（ADH）与 NAD 和底物类似物 2,2,2-三氟乙醇复合物中的催化锌与来自蛋白质的两个半胱氨酸残基和一个组氨酸残基以及来自醇的氧配位。锌促进醇的去质子化，对催化至关重要。在酵母脱辅基酶中，锌与附近的谷氨酸配位，在与 NAD 的复合物中，谷氨酸被醇取代。一些同源的中链脱氢酶具有一个半胱氨酸被天冬氨酸或谷氨酸残基取代。通过使用定点突变将酿酒酵母 ADH1 中的 Cys-153 替换为天冬氨酸来研究天冬氨酸如何影响催化作用。如果缓冲液中不包含 EDTA，则 C153D 酶与野生型酶一样稳定。该取代物使 NAD 的亲和力增加了 3 倍，但不影响 NADH 结合。在 pH 7.3 时，乙醇氧化的周转率（V/E）降低了 7 倍，催化效率降低了 18 倍（V/EK），但乙醛还原的周转率（V/E）与野生型酶相同，催化效率降低了 8 倍（V/EK）。乙醇氧化的 V/E 和 V/EK 的氘同位素效应分别为 3.0 和 3.8，表明对于 C153D 酶，氢化物转移对于周转率的限制比对野生型酶更重要。对于两种酶，V/EK 对乙醇氧化的 pH 依赖性模式在 pH 7 到 9 的范围内相似。C153D 取代使三氟乙醇的结合降低了 5 倍，使吡唑的结合降低了 65 倍。C153D 和野生型 ADH 对伯醇的底物特异性具有相似的模式。仲醇的效率仅降低了约 4 倍，而 1,2-丙二醇和丙酮的效率与野生型酶大致相同。C153D 取代通过改变锌上的配体交换或局部结构，适度影响催化作用。综述并分析了具有同源半胱氨酸取代的相关中链脱氢酶的酸碱催化结构和机制。
