Ehrig T, Bohren K M, Prendergast F G, Gabbay K H
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905.
Biochemistry. 1994 Jun 14;33(23):7157-65. doi: 10.1021/bi00189a019.
Aldose reductase enfolds NADP+/NADPH via a complex loop mechanism, with cofactor exchange being the rate-limiting step for the overall reaction. This study measures the binding constants of these cofactors in the wild-type enzyme, as well as a variety of active-site mutants (C298A, Y48H, Y48F, Y209F, H110A, W219A, and W20A), and seeks to identify the binding site and mechanism of the aldose reductase inhibitor alrestatin in the recombinant human enzyme. All the mutant enzymes, regardless of their enzyme activities, have normal or only slightly elevated coenzyme binding constants, suggesting a tertiary structure similar to that of the wild-type enzyme. Binding of alrestatin was detected by fluorescence assays, and by an ultrafiltration assay which measures the fraction of unbound alrestatin. Alrestatin binds preferentially to the enzyme/NADP+ complex, consistent with the steady-state inhibition pattern. Alrestatin binding and enzyme inhibition were abolished in the Tyr48 mutants Y48F and Y48H, implicating the positively charged anion well formed by the Asp43-/Lys77+/Tyr48(0)/NADP+ complex in inhibitor binding (Harrison et al., 1994; Bohren et al., 1994). The enzyme mutant W20A severely affected the inhibitory potencies of a variety of commercially developed aldose reductase inhibitors (zopolrestat, tolrestat, FK366, AL1576, alrestatin, ponalrestat, and sorbinil). Inhibition by citrate, previously shown to bind to the positively charged anion well, was not affected by this mutation. Inhibitors with flexible double aromatic ring systems (Zopolrestat, FK366, and ponalrestat) were less affected than others possessing a single aromatic ring system, suggesting that the additional pharmacophor ring system stabilizes the inhibitor by interaction at some other hydrophobic site.(ABSTRACT TRUNCATED AT 250 WORDS)
醛糖还原酶通过一种复杂的环机制折叠NADP⁺/NADPH，辅因子交换是整个反应的限速步骤。本研究测量了这些辅因子在野生型酶以及多种活性位点突变体（C298A、Y48H、Y48F、Y209F、H110A、W219A和W20A）中的结合常数，并试图确定重组人酶中醛糖还原酶抑制剂阿雷司他汀的结合位点和机制。所有突变酶，无论其酶活性如何，都具有正常或仅略有升高的辅酶结合常数，表明其三级结构与野生型酶相似。通过荧光测定法以及测量未结合阿雷司他汀分数的超滤测定法检测阿雷司他汀的结合。阿雷司他汀优先结合酶/NADP⁺复合物，这与稳态抑制模式一致。在Tyr48突变体Y48F和Y48H中，阿雷司他汀的结合和酶抑制作用被消除，这表明由Asp43⁻/Lys77⁺/Tyr48(0)/NADP⁺复合物形成的带正电荷的阴离子阱在抑制剂结合中起作用（哈里森等人，1994年；博伦等人，1994年）。酶突变体W20A严重影响了多种商业开发的醛糖还原酶抑制剂（唑泊司他、托瑞司他、FK366、AL1576、阿雷司他汀、泊那司他和索比尼尔）的抑制效力。先前显示与带正电荷的阴离子阱结合的柠檬酸盐的抑制作用不受此突变影响。具有柔性双芳香环系统的抑制剂（唑泊司他、FK366和泊那司他）受到的影响小于具有单芳香环系统的其他抑制剂，这表明额外的药效团环系统通过在其他一些疏水位点的相互作用使抑制剂稳定。（摘要截短于250字）
