Barski O A, Gabbay K H, Grimshaw C E, Bohren K M
Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030, USA.
Biochemistry. 1995 Sep 5;34(35):11264-75. doi: 10.1021/bi00035a036.
Human aldehyde reductase is a NADPH-dependent aldo-keto reductase that is closely related (65% identity) to aldose reductase, an enzyme involved in the pathogenesis of some diabetic and galactosemic complications. In aldose reductase, the active site residue Tyr48 is the proton donor in a hydrogen-bonding network involving residues Asp43/Lys77, while His110 directs the orientation of substrates in the active site pocket. Mutation of the homologous Tyr49 to phenylalamine or histidine (Y49F or Y49H) and of Lys79 to methionine (K79M) in aldehyde reductase yields inactive enzymes, indicating similar roles for these residues in the catalytic mechanism of aldehyde reductase. A H112Q mutant aldehyde reductase exhibited a substantial decrease in catalytic efficiency (kcat/Km) for hydrophilic (average 150-fold) and aromatic substrates (average 4200-fold) and 50-fold higher IC50 values for a variety of inhibitors than that of the wild-type enzyme. The data suggest that His112 plays a major role in determining the substrate specificity of aldehyde reductase, similar to that shown earlier for the homologous His110 in aldose reductase [Bohren, K. M., et. al. (1994) Biochemistry 33, 2021-2032]. Mutation of Ile298 or Val299 affected the kinetic parameters to a much lesser degree. Unlike native aldose reductase, which contains a thiol-sensitive Cys298, neither the I298C or V299C mutant exhibited any thiol sensitivity, suggesting a geometry of the active site pocket different from that in aldose reductase. Also different from aldose reductase, the detection of a significant primary deuterium isotope effect on kcat (1.48 +/- 0.02) shows that nucleotide exchange is only partially rate-limiting. Primary substrate and solvent deuterium isotope effects on the H112Q mutant suggest that hydride and proton transfers occur in two discrete steps with hydride transfer taking place first. Dissociation constants and spectroscopic and fluorimetric properties of nucleotide complexes with various mutants suggest that, in addition to Tyr49 and His112, Lys79 plays a hitherto unappreciated role in nucleotide binding. The mode of inhibition of aldehyde reductase by aldose reductase inhibitors (ARIs) is generally similar to that of aldose reductase and involves binding to the E:NADP+ complex, as shown by kinetic and direct inhibitor-binding experiments. The order of ARI potency was AL1576 (Ki = 60 nM) > tolrestat > ponalrestat > sorbinil > FK366 > zopolrestat > alrestatin (Ki = 148 microM). Our data on aldehyde reductase suggest that the active site pocket significantly differs from that of aldose reductase, possibly due to the participation of the C-terminal loop in its formation.
人醛还原酶是一种依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的醛酮还原酶，与醛糖还原酶密切相关（同一性为65%），醛糖还原酶是一种参与某些糖尿病和半乳糖血症并发症发病机制的酶。在醛糖还原酶中，活性位点残基Tyr48是涉及Asp43/Lys77残基的氢键网络中的质子供体，而His110则指导底物在活性位点口袋中的取向。醛还原酶中同源的Tyr49突变为苯丙氨酸或组氨酸（Y49F或Y49H）以及Lys79突变为甲硫氨酸（K79M）会产生无活性的酶，表明这些残基在醛还原酶的催化机制中具有相似作用。一个H112Q突变型醛还原酶对亲水性底物（平均降低150倍）和芳香族底物（平均降低4200倍）的催化效率（kcat/Km）大幅下降，并且对于多种抑制剂的半数抑制浓度（IC50）值比野生型酶高50倍。数据表明，His112在决定醛还原酶的底物特异性方面起主要作用，类似于醛糖还原酶中同源的His110之前所显示的作用[博伦，K.M.等人（1994年）《生物化学》33卷，2021 - 2032页]。Ile298或Val299的突变对动力学参数的影响程度要小得多。与含有对硫醇敏感的Cys298的天然醛糖还原酶不同，I298C或V299C突变体均未表现出任何硫醇敏感性，这表明活性位点口袋的几何结构与醛糖还原酶不同。同样与醛糖还原酶不同的是，对kcat检测到显著的一级氘同位素效应（1.48±0.02）表明核苷酸交换只是部分限速步骤。对H112Q突变体的一级底物和溶剂氘同位素效应表明，氢化物和质子转移分两个离散步骤进行，氢化物转移先发生。各种突变体与核苷酸复合物的解离常数以及光谱和荧光性质表明，除了Tyr49和His112外，Lys79在核苷酸结合中起着迄今未被重视的作用。醛糖还原酶抑制剂（ARIs）对醛还原酶的抑制模式通常与醛糖还原酶相似，并且涉及与E:NADP + 复合物结合，动力学和直接抑制剂结合实验表明了这一点。ARI效力顺序为AL1576（Ki = 60 nM）>托瑞司他>泊那司他>索比尼尔>FK366>唑泊司他>阿雷司他（Ki = 148 microM）。我们关于醛还原酶的数据表明，活性位点口袋与醛糖还原酶的显著不同，这可能是由于C末端环参与其形成所致。
