Studies on the mechanism of action of xanthine oxidase
Studies on the mechanism of action of xanthine oxidase
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DOI：
10.1016/j.jinorgbio.2003.11.010
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发表时间：
2004-05-01
影响因子：
3.9
通讯作者：
Hille, R
中科院分区：
文献类型：
--
作者：
Choi, EY;Stockert, AL;Hille, R
本文介绍了近年来对含铼的黄嘌呤氧化酶的反应机理的研究。该酶的稳态和快速反应动力学的pH依赖性均呈钟形，酸性和碱性分支的pK（a）s分别为6.6和7.4。这些被分配到一个活性位点的碱和底物的电离，分别与暗示，酶的嘌呤底物的中性，而不是单阴离子形式的行为。一个计算研究提供的证据表明，在反应过程中发生底物的互变异构化，与质子从N-3移动到N-9的反应过程中，酶促进这种互变异构化可能有助于多达24千卡/摩尔的过渡态稳定的反应。电子自旋回波（ESEEM）和电子-核双共振（ENDOR）研究所谓的“非常快速”的Mo（V）反应中间体，后者使用新合成的底物2-羟基-6-甲基嘌呤（已在C-8处选择性同位素标记），表明产物以简单的末端方式与活性位点的钼结合，与涉及（去质子化的）Mo-OH对底物的C-8位的亲核攻击的反应机理一致。使用一系列嘌呤的动力学研究未能确定底物的单电子还原电位和催化有效性之间的相关性，表明由单电子外层电子转移引发的反应机制是不可能的。最后，在上述工作的背景下，活性位点结构的考虑建议定点诱变研究靶向的特定氨基酸残基。两个这样的突变体的初步实验是完全一致的两个活性位点谷氨酸残基的催化作用。(C)2003年爱思唯尔公司All rights reserved.
Recent studies of the reaction mechanism of the molybdenum-containing enzyme xanthine oxidase are presented. The pH-dependence of both the steady-state and rapid reaction kinetics of the enzyme exhibits is bell-shaped, with pK(a)s for the acid and alkaline limbs of 6.6 and 7.4, respectively. These are assigned to ionizations of an active site base and substrate, respectively, with the implication that enzyme acts on the neutral rather than monoanionic form of the purine substrate. A computational study provides evidence that in the course of the reaction tautomerization of substrate occurs, with a proton moving from N-3 to N-9 in the course of the reaction-enzyme facilitation of this tautomerization may contribute as much as 24 kcal/mol in transition state stabilization for the reaction. Electron spin echo (ESEEM) and electron-nuclear double resonance (ENDOR) studies of the so-called "very rapid" Mo(V) intermediate of the reaction, the latter work using a newly synthesized form of the substrate 2-hydroxy-6-methylpurine that has been selectively isotopically labeled at C-8, indicates that product is bound to the molybdenum of the active site in a simple, end-on fashion, consistent with a reaction mechanism involving nucleophilic attack of a (deprotonated) Mo-OH on the C-8 position of substrate. A kinetic study using a series of purines has failed to identify a correlation between the one-electron reduction potential for substrate and catalytic effectiveness, indicating that a reaction mechanism initiated by one-electron, outer-sphere electron transfer is unlikely. Finally, a consideration of the active site structure in the context of the above work suggests specific amino acid residues to target for site-directed mutagenesis studies. Preliminary experiments with two such mutants are entirely consistent with the proposed catalytic roles of two active site glutamate residues. (C) 2003 Elsevier Inc. All rights reserved.