Harrison D H, Bohren K M, Ringe D, Petsko G A, Gabbay K H
Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.
Biochemistry. 1994 Mar 1;33(8):2011-20. doi: 10.1021/bi00174a006.
Aldose reductase is a NADPH-dependent aldo-keto reductase involved in the pathogenesis of some diabetic and galactosemic complications. The published crystal structure of human aldose reductase [Wilson et al. (1992) Science 257, 81-84] contains a hitherto unexplained electron density positioned within the active site pocket facing the nicotinamide ring of the NADPH and other key active site residues (Tyr48, His110, and Cys298). In this paper we identify the electron density as citrate, which is present in the crystallization buffer (pH 5.0), and provide confirmatory evidence by both kinetic and crystallographic experiments. Citrate is an uncompetitive inhibitor in the forward reaction with respect to aldehyde (reduction of aldehyde), while it is a competitive inhibitor with respect to alcohol in the backward reaction (oxidation of alcohol), indicating that it interacts with the enzyme-NADP(+)-product complex. Citrate can be replaced in the crystalline enzyme complex by cacodylate or glucose 6-phosphate; the structure of each of these complexes shows the specific molecule bound in the active site. All of the structures have been determined to a nominal resolution of 1.76 A and refined to R-factors below 18%. While cacodylate can be bound within the active site under the crystallization conditions, it does not inhibit the wild-type enzyme in solution. Glucose 6-phosphate, however, is a substrate for aldose reductase. The similar location of the negative charges of citrate, cacodylate, and glucose 6-phosphate within the active site suggests an anion-binding site delineated by the C4N of nicotinamide, the OH of Tyr48, and the N epsilon of His110. The location of citrate binding in the active site leads to a plausible catalytic mechanism for aldose reductase.
醛糖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，参与某些糖尿病和半乳糖血症并发症的发病机制。已发表的人醛糖还原酶晶体结构[威尔逊等人（1992年），《科学》257卷，81 - 84页]在活性位点口袋内包含一个迄今无法解释的电子密度，该口袋面向NADPH的烟酰胺环和其他关键活性位点残基（Tyr48、His110和Cys298）。在本文中，我们确定该电子密度为柠檬酸盐，它存在于结晶缓冲液（pH 5.0）中，并通过动力学和晶体学实验提供了确证证据。柠檬酸盐在醛（醛的还原）的正向反应中是一种非竞争性抑制剂，而在反向反应（醇的氧化）中相对于醇是一种竞争性抑制剂，这表明它与酶 - NADP(+) - 产物复合物相互作用。在晶体酶复合物中，柠檬酸盐可以被二甲胂酸盐或6 - 磷酸葡萄糖取代；这些复合物中每一个的结构都显示了结合在活性位点的特定分子。所有结构的测定名义分辨率为1.76埃，并精修至R因子低于18%。虽然在结晶条件下二甲胂酸盐可以结合在活性位点内，但它在溶液中不抑制野生型酶。然而，6 - 磷酸葡萄糖是醛糖还原酶的底物。柠檬酸盐、二甲胂酸盐和6 - 磷酸葡萄糖在活性位点内负电荷的相似位置表明，一个由烟酰胺的C4N、Tyr48的OH和His110的Nε界定的阴离子结合位点。柠檬酸盐在活性位点的结合位置导致了醛糖还原酶一种合理的催化机制。
