Grimshaw C E, Bohren K M, Lai C J, Gabbay K H
Whittier Diabetes Program, Department of Medicine, University of California, San Diego, La Jolla 92093-0983, USA.
Biochemistry. 1995 Nov 7;34(44):14356-65. doi: 10.1021/bi00044a012.
We have used transient kinetic data for partial reactions of recombinant human aldose reductase and simulations of progress curves for D-xylose reduction with NADPH and for xylitol oxidation with NADP+ to estimate rate constants for the following mechanism at pH 8.0: E<-->E.NADPH<-->*E.NADPH<-->*E.NADPH.RCHO<-->*E.NADP+.RCH2OH <-->*E.NADP+<--> E.NADP+<-->E. The mechanism includes kinetically significant conformational changes of the two binary E.nucleotide complexes which correspond to the movement of a crystallographically identified nucleotide-clamping loop involved in nucleotide exchange. The magnitude of this conformational clamping is substantial and results in a 100- and 650-fold lowering of the nucleotide dissociation constant in the productive *E.NADPH and *E.NADP+ complexes, respectively. The transient reduction of D-xylose displays burst kinetics consistent with the conformational change preceding NADP+ release (*E.NADP+-->E.NADP+) as the rate-limiting step in the forward direction. The maximum burst rate also displays a large deuterium isotope effect (Dkburst = 3.6-4.1), indicating that hydride transfer contributes significantly to rate limitation of the sequence of steps up to and including release of xylitol. In the reverse reaction, no burst of NADPH production is observed because the hydride transfer step is overall 85% rate-limiting. Even so, the conformational change preceding NADPH release (*E.NADPH-->E.NADPH) still contributes 15% to the rate limitation for reaction in this direction. The estimated rate constant for hydride transfer from NADPH to the aldehyde of D-xylose (130 s-1) is only 5- to 10-fold lower than the corresponding rate constant determined for NADH-dependent carbonyl reduction catalyzed by lactate or liver alcohol dehydrogenase. Hydride transfer from alcohol to NADP+ (0.6 s-1), however, is at least 100- to 1000-fold slower than NAD(+)-dependent alcohol oxidation mediated by these two enzymes, resulting in a bound-state equilibrium constant for aldose reductase which greatly favors the forward reaction. The proposed kinetic model provides a basic set of rate constants for interpretation of kinetic results obtained with aldose reductase mutants generated for the purpose of examining structure-function relationships of different components of the native enzyme.
我们利用重组人醛糖还原酶部分反应的瞬态动力学数据，以及用NADPH还原D-木糖和用NADP⁺氧化木糖醇的进程曲线模拟，来估算pH 8.0时以下机制的速率常数：E⇌E·NADPH⇌*E·NADPH⇌*E·NADPH·RCHO⇌E·NADP⁺·RCH₂OH⇌E·NADP⁺⇌E·NADP⁺⇌E。该机制包括两个二元E-核苷酸复合物在动力学上显著的构象变化，这与晶体学鉴定的参与核苷酸交换的核苷酸钳位环的移动相对应。这种构象钳制的程度很大，分别导致在有活性的E·NADPH和E·NADP⁺复合物中核苷酸解离常数降低100倍和650倍。D-木糖的瞬态还原显示出爆发动力学，这与NADP⁺释放（*E·NADP⁺→E·NADP⁺）之前的构象变化一致，是正向反应中的限速步骤。最大爆发速率也显示出较大的氘同位素效应（Dkburst = 3.6 - 4.1），表明氢化物转移对直至并包括木糖醇释放的一系列步骤的速率限制有显著贡献。在逆向反应中，未观察到NADPH产生的爆发，因为氢化物转移步骤总体上是85%的限速步骤。即便如此，NADPH释放（*E·NADPH→E·NADPH）之前的构象变化仍对该方向反应的速率限制贡献15%。从NADPH到D-木糖醛基的氢化物转移的估算速率常数（130 s⁻¹）仅比乳酸或肝醇脱氢酶催化的依赖NADH的羰基还原所确定的相应速率常数低5至10倍。然而，从醇到NADP⁺的氢化物转移（0.6 s⁻¹）比这两种酶介导的依赖NAD⁺的醇氧化至少慢100至1000倍，导致醛糖还原酶的结合态平衡常数极大地有利于正向反应。所提出的动力学模型提供了一组基本的速率常数，用于解释为研究天然酶不同组分的结构-功能关系而产生的醛糖还原酶突变体的动力学结果。
