Barski O A, Gabbay K H, Bohren K M
Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030, USA.
Biochemistry. 1996 Nov 12;35(45):14276-80. doi: 10.1021/bi9619740.
Human aldehyde reductase has a preference for carboxyl group-containing negatively charged substrates. It belongs to the NADPH-dependent aldo-keto reductase superfamily whose members are in part distinguished by unique C-terminal loops. To probe the role of the C-terminal loops in determining substrate specificities in these enzymes, two arginine residues, Arg308 and Arg311, located in the C-terminal loop of aldehyde reductase, and not found in any other C-terminal loop, were replaced with alanine residues. The catalytic efficiency of the R311A mutant for aldehydes containing a carboxyl group is reduced 150-250-fold in comparison to that of the wild-type enzyme, while substrates not containing a negative charge are unaffected. The R311A mutant is also significantly less sensitive to inhibition by dicarboxylic acids, indicating that Arg311 interacts with one of the carboxyl groups. The inhibition pattern indicates that the other carboxyl group binds to the anion binding site formed by Tyr49, His112, and the nicotinamide moiety of NADP+. The correlation between inhibitor potency and the length of the dicarboxylic acid molecules suggests a distance of approximately 10 A between the amino group of Arg311 and the anion binding site in the aldehyde reductase molecule. The sensitivity of inhibition of the R311A mutant by several commercially available aldose reductase inhibitors (ARIs) was variable, with tolrestat and zopolrestat becoming more potent inhibitors (30- and 5-fold, respectively), while others remained the same or became less potent. The catalytic properties, substrate specificity, and susceptibility to inhibition of the R308A mutant remained similar to that of the wild-type enzyme. The data provide direct evidence for C-terminal loop participation in determining substrate and inhibitor specificity of aldo-keto reductases and specifically identifies Arg311 as the basis for the carboxyl-containing substrate preference of aldehyde reductase.
人醛还原酶偏好含羧基的带负电荷底物。它属于依赖NADPH的醛酮还原酶超家族，其成员部分通过独特的C末端环来区分。为了探究C末端环在决定这些酶底物特异性中的作用，位于醛还原酶C末端环且在其他C末端环中未发现的两个精氨酸残基Arg308和Arg311被丙氨酸残基取代。与野生型酶相比，R311A突变体对含羧基醛的催化效率降低了150 - 250倍，而不含负电荷的底物不受影响。R311A突变体对二羧酸抑制的敏感性也显著降低，表明Arg311与其中一个羧基相互作用。抑制模式表明另一个羧基与由Tyr49、His112和NADP + 的烟酰胺部分形成的阴离子结合位点结合。抑制剂效力与二羧酸分子长度之间的相关性表明，醛还原酶分子中Arg311的氨基与阴离子结合位点之间的距离约为10埃。几种市售醛糖还原酶抑制剂（ARIs）对R311A突变体抑制的敏感性各不相同，托瑞司他和唑泊司他成为更强效的抑制剂（分别为30倍和5倍），而其他抑制剂效力保持不变或降低。R308A突变体的催化特性、底物特异性和抑制敏感性与野生型酶相似。这些数据为C末端环参与决定醛酮还原酶的底物和抑制剂特异性提供了直接证据，并特别确定Arg311是醛还原酶偏好含羧基底物的基础。
