Department of Biochemistry, The University of Iowa, Iowa City, IA 52242-1109, USA.
Chem Biol Interact. 2011 May 30;191(1-3):42-7. doi: 10.1016/j.cbi.2010.12.015. Epub 2010 Dec 22.
The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 ("stomach" isoenzyme) are substantially larger (10-100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class I enzymes is substituted with Cys in human ADH4, and locally different conformations of the peptide backbones could affect coenzyme binding. This hypothesis was tested by making the A317C substitution in horse liver ADH1E and comparisons to the wild-type ADH1E. The steady-state kinetic constants for the oxidation of benzyl alcohol and the reduction of benzaldehyde catalyzed by the A317C enzyme were very similar (up to about 2-fold differences) to those for the wild-type enzyme. Transient kinetics showed that the rate constants for binding of NAD(+) and NADH were also similar. Transient reaction data were fitted to the full Ordered Bi Bi mechanism and showed that the rate constants for hydride transfer decreased by about 2.8-fold with the A317C substitution. The structure of A317C ADH1E complexed with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol at 1.2 Å resolution is essentially identical to the structure of the wild-type enzyme, except near residue 317 where the additional sulfhydryl group displaces a water molecule that is present in the wild-type enzyme. ADH is adaptable and can tolerate internal substitutions, but the protein dynamics apparently are affected, as reflected in rates of hydride transfer. The A317C substitution is not solely responsible for the larger kinetic constants in human ADH4; thus, the differences in catalytic activity must arise from one or more of the other hundred substitutions in the enzyme.
人醇脱氢酶（ADH）4（“胃”同工酶）的周转率和其他动力学常数比人 I 类和马肝醇脱氢酶大得多（10-100 倍）。对这些酶的一级氨基酸序列（69%的同源性）和三级结构的比较表明，与辅酶的烟酰胺酰胺氮形成氢键的残基 317 可能是造成这些差异的原因。I 类酶中的丙氨酸 317 被人 ADH4 中的半胱氨酸取代，肽骨架的局部不同构象可能会影响辅酶结合。通过在马肝醇脱氢酶 1E 中进行 A317C 取代并与野生型 ADH1E 进行比较，对该假设进行了测试。A317C 酶催化苄醇氧化和苯甲醛还原的稳态动力学常数非常相似（差异高达 2 倍）。瞬态动力学表明，NAD（+）和 NADH 结合的速率常数也相似。瞬态反应数据拟合为完全有序双酶双机制，表明氢化物转移的速率常数随着 A317C 取代降低了约 2.8 倍。分辨率为 1.2Å 的 A317C ADH1E 与 NAD（+）和 2,3,4,5,6-五氟苄醇复合物的结构与野生型酶基本相同，除了残基 317 附近，额外的巯基取代了存在于野生型酶中的一个水分子。ADH 具有适应性，可以容忍内部取代，但显然蛋白质动力学受到影响，这反映在氢化物转移的速率上。A317C 取代并不是人 ADH4 中较大动力学常数的唯一原因；因此，催化活性的差异必须来自酶的其他一百个取代中的一个或多个。
