De Winter H L, von Itzstein M
Department of Medicinal Chemistry, Victorian College of Pharmacy, Monash University, Parkville, Australia.
Biochemistry. 1995 Jul 4;34(26):8299-308. doi: 10.1021/bi00026a011.
In an attempt to obtain a picture of the binding conformation of aldehyde substrates to human aldose reductase (hAR), modeling calculations have been performed on the binding of three substrates, D-xylose, L-xylose, and D-lyxose, to wild-type human aldose reductase and two of its site-directed mutants. It was found that the average geometry of D-xylose in the active site of wild-type aldose reductase is characterized by strong hydrogen bonds involving the reactive carbonyl oxygen of the substrate and both Tyr48 and His110. The calculations also suggest the importance of Trp111 in the binding of 2'-hydroxyl-containing aldehyde substrates. A good correlation between calculated interaction enthalpies and experimental log(Km) or log(kcat/Km) values was obtained when His110 was modeled with its N epsilon 2 atom protonated and N delta 1 unprotonated. No correlation was found for the other two configurations of His110. On the basis of comparisons of the calculated substrate binding conformations for the three possible His110 configurations, and on the correlations between measured log(Km) or log(kcat/Km) and calculated parameters, it is proposed that His110 is neutral and protonated at N epsilon 2 when an aldehyde substrate is bound to the hAR/NADPH complex. A chain of three hydrogen-bonded water molecules has been identified in all available crystal structures and is located in an enzyme channel which links the N delta 1 atom of His110 to the solvent-accessible surface of the enzyme. A possible role of this channel in the mechanism of catalysis of aldose reductase is suggested.
为了获得醛底物与人醛糖还原酶（hAR）结合构象的图像，已对三种底物D-木糖、L-木糖和D-来苏糖与野生型人醛糖还原酶及其两个定点突变体的结合进行了建模计算。结果发现，野生型醛糖还原酶活性位点中D-木糖的平均几何结构的特征是，底物的反应性羰基氧与Tyr48和His110之间形成了强氢键。计算还表明，Trp111在含2'-羟基醛底物的结合中具有重要作用。当His110的Nε2原子质子化而Nδ1未质子化时，计算得到的相互作用焓与实验log（Km）或log（kcat/Km）值之间具有良好的相关性。对于His110的其他两种构型，未发现相关性。基于对His110三种可能构型计算得到的底物结合构象的比较，以及测量的log（Km）或log（kcat/Km）与计算参数之间的相关性，提出当醛底物与hAR/NADPH复合物结合时，His110在Nε2处呈中性且质子化。在所有可用的晶体结构中都发现了由三个氢键连接的水分子链，其位于一个将His110的Nδ1原子与酶的溶剂可及表面相连的酶通道中。本文提出了该通道在醛糖还原酶催化机制中的可能作用。
