Catalytic reaction profile for NADH-dependent reduction of aromatic aldehydes by xylose reductase from Candida tenuis
Catalytic reaction profile for NADH-dependent reduction of aromatic aldehydes by xylose reductase from Candida tenuis
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DOI：
10.1042/bj20020080
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发表时间：
2002-09-15
影响因子：
4.1
通讯作者：
Nidetzky, B
中科院分区：
文献类型：
--
作者：
Mayr, P;Nidetzky, B
动力学取代基效应已用于检查来自酵母细念珠菌的木糖还原酶的催化反应谱，木糖还原酶是初级碳水化合物代谢的代表性醛/酮还原酶。 NADH 依赖性酶促醛还原的 Michaelis-Menten 参数（k(cat) 和 K-m）已使用一系列同源苯甲醛衍生物测定，其中使用间位和对位取代基来系统地干扰反应性羰基的性质。通过使用在 pro-R C4-H 位上标记的 NADH H-2 获得了间位取代苯甲醛酶反应中 k(cat) 和 k(cat)/K-m 的动力学同位素效应 (KIE)，并在 NAD(H) 和酶存在下测量了这些醛转化为相应醇 (K-eq) 的平衡常数。醛解离常数 (K-d) 和氢化物转移速率常数 (k(7)) 是根据稳态速率和 KIE 数据计算得出的。定量结构-活性关系分析用于将观察到的 k(cat)/K-m 取代基依赖性分解为主要电子效应和对位取代基的生产位置效应。 k(cat)/K-m（取代基位置校正后）和 K-eq 遵循取代基参数 Hammett sigma 的对数线性相关性，给出 + 1.4 至 + 1.7 的相同斜率值 (rho)，而 log K-d 的相同 Hammett 图得出 rho = -1.5。由此得出的结论是，吸电子取代基促进反应并在大约相同程度上增加结合。 k(cat) (1.8) 和 k(cat)/K-m (2.7) 的 KIE 值以及 k(7) 同样显示出不依赖于取代基。因此，无论所研究的底物系列上观察到的反应性变化如何，氢化物转移的限速过渡态的特征都没有发生变化。 rho 值的符号和大小表明，就活性碳的电荷发展而言，这种过渡态是类似产品的。结构-反应性相关性揭示了 NADPH 特异性和双 NADPH/NADH 特异性酵母木糖还原酶之间以及两个醛/酮还原酶家族之间的活性位点同源性，尽管产生木糖还原酶（家族 2B）和醛还原酶（家族 1A）的宿主生物体存在系统发育分离。
Kinetic substituent effects have been used to examine the catalytic reaction profile of xylose reductase from the yeast Candida tenuis, a representative aldo/keto reductase of primary carbohydrate metabolism. Michaelis-Menten parameters (k(cat) and K-m) for NADH-dependent enzymic aldehyde reductions have been determined using a homologous series of benzaldehyde derivatives in which substituents in meta and para positions were employed to systematically perturb the properties of the reactive carbonyl group. Kinetic isotope effects (KIEs) on k(cat) and k(cat)/K-m for enzymic reactions with meta-substituted benzaldehydes have been obtained by using NADH H-2-labelled in the pro-R C4-H position, and equilibrium constants for the conversion of these aldehydes into the corresponding alcohols (K-eq) have been measured in the presence of NAD(H) and enzyme. Aldehyde dissociation constants (K-d) and the hydride transfer rate constant (k(7)) have been calculated from steady-state rate and KIE data. Quantitative structure-activity relationship analysis was used to factor the observed substituent dependence of k(cat)/K-m into a major electronic effect and a productive positional effect of the para substituent. k(cat)/K-m (after correction for substituent position) and K-eq obeyed log-linear correlations over the substituent parameter, Hammett sigma, giving identical slope values (rho) of + 1.4 to + 1.7, whereas the same Hammett plot for log K-d yielded rho = -1.5. This leads to the conclusion that electron-withdrawing substituents facilitate the reaction and increase binding to about the same extent. KIE values for k(cat) (1.8) and k(cat)/K-m (2.7), and likewise k(7), showed no substituent dependence. Therefore, irrespective of the observed changes in reactivity over the substrate series studied no shift in the character of the rate-limiting transition state of hydride transfer occurred. The signs and magnitudes of rho values suggest this transition state to be product-like in terms of charge development at the reactive carbon. Structure-reactivity correlations reveal active-site homologies among NADPH-specific and dual NADPH/NADH-specific yeast xylose reductases and across two aldo/keto reductase families in spite of the phylogenetic separation of the host organisms producing xylose reductase (family 2B) and aldehyde reductase (family 1A).