HUMAN ALDOSE REDUCTASE - PK OF TYROSINE-48 REVEALS THE PREFERRED IONIZATION STATE FOR CATALYSIS AND INHIBITION
HUMAN ALDOSE REDUCTASE - PK OF TYROSINE-48 REVEALS THE PREFERRED IONIZATION STATE FOR CATALYSIS AND INHIBITION
复制标题
DOI：
10.1021/bi00044a014
复制
发表时间：
1995-11-07
期刊：
影响因子：
2.9
通讯作者：
GABBAY, KH
中科院分区：
文献类型：
--
作者：
GRIMSHAW, CE;BOHREN, KM;GABBAY, KH
详细分析了重组人醛糖还原酶介导的正向醛还原和反向醇氧化反应的动力学参数的pH变化，用于抑制剂结合，以及对醛还原的动力学同位素效应的研究表明，活性位点酸-碱催化剂基团Tyr 48的pK值对结合核苷酸的氧化态非常敏感（NADPH或NADP（+））和Cys 298巯基部分的存在或不存在。因此，C298 A突变酶的Tyr 48残基在生产性 *E中显示出范围为7.6的pK值。NADP（+）复合物，结合并与醇类反应至生产性 *E中的8.7。与醛底物结合并反应的NADPH复合物。对于野生型酶，后一种复合物中的Tyr 48显示出约8.25的较低pK值。通过识别和定量正向反应中几种醛底物的粘性程度，促进了pK值的分配。V-醛/E（t）和V-D（醛）的不寻常的pH依赖性，通过一个波降低约20倍，并在高pH值下保持恒定，分别显示来自NADP（+）释放的净速率的pH依赖性降低。所描述的结果与先前提出的醛糖还原酶催化的化学机理完全一致（Bohren等人，1994），并进一步确定了螺乙内酰脲类醛糖还原酶抑制剂，例如，sorbinil通过反向质子化方案发生，其中离子化的抑制剂优先与 *E结合。NADP（+）与Tyr 48的复合物以质子化羟基形式存在。后者的发现使我们能够提出一个统一的模型，高亲和力的醛糖还原酶抑制剂结合，重点是过渡态样性质的 * E-Tyr 48-OH。NADP（+）。抑制剂（-）络合物。
Detailed analyses of the pH variation of kinetic parameters for the forward aldehyde reduction and reverse alcohol oxidation reactions mediated by recombinant human aldose reductase, for inhibitor binding, and for kinetic isotope effects on aldehyde reduction have revealed that the pK value for the active site acid-base catalyst group Tyr48 is quite sensitive to the oxidation state of the bound nucleotide (NADPH or NADP(+)) and to the presence or absence of the Cys298 sulfhydryl moiety. Thus, the Tyr48 residue of C298A mutant enzyme displays a pK value that ranges from 7.6 in the productive *E . NADP(+) complex that binds and reacts with alcohols to 8.7 in the productive *E . NADPH complex that binds and reacts with aldehyde substrates. For wild-type enzyme, Tyr48 in the latter complex displays a lower pK value of about 8.25. Assignment of the pK values was facilitated by the recognition and quantitation of the degree of stickiness of several aldehyde substrates in the forward reaction. The unusual pH dependence for V-aldehyde/E(t) and V-D(aldehyde), which decrease roughly 20-fold through a wave and remain constant at high pH, respectively, is shown to arise from the pH-dependent decrease in the net rate of NADP(+) release. The results described are fully consistent with the chemical mechanism for aldose reductase catalysis proposed previously (Bohren et al., 1994) and, furthermore, establish that binding of the spirohydantoin class of aldose reductase inhibitors, e.g., sorbinil, occurs via a reverse protonation scheme in which the ionized inhibitor binds preferentially to the *E . NADP(+) complex with Tyr48 present as the protonated hydroxyl form. The latter finding allows us to propose a unified model for high-affinity aldose reductase inhibitor binding that focuses on the transition state-like nature of the *E-Tyr48-OH . NADP(+) . inhibitor(-)complex.