The oxidative half-reaction of xanthine dehydrogenase with NAD; Reaction kinetics and steady-state mechanism
The oxidative half-reaction of xanthine dehydrogenase with NAD; Reaction kinetics and steady-state mechanism
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DOI：
10.1074/jbc.272.45.28335
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发表时间：
1997-11-07
影响因子：
4.8
通讯作者：
Massey, V
中科院分区：
文献类型：
--
作者：
Harris, CM;Massey, V
研究了牛乳中还原型黄嘌呤脱氢酶（XDH）与NAD的反应。理解这个反应是必要的XDH营业额与其假定的天然电子受体的完整描述，并解决的偏好WH NAD超过氧作为底物。用停流光度法研究了预还原XDH与NAD的反应。发现该反应涉及用2当量NAD的两轮氧化。第一轮完成，第二轮达到稍微不利的均衡。NAD快速结合，表观Kd为25 +/- 2 μ M，随后NAD以130 +/- 13 s（-1）的速率常数还原。NADH以42 +/- 12 s（-1）的速率常数解离完成一轮氧化。这些步骤已经成功地进行了测试和建模，在第二轮氧化中重复进行。据估计，NAD结合的缔合速率常数远大于在分子氧氧化中测得的任何速率常数，从而解释了NAD如何与氧竞争还原当量。NAD还原和NADH解离的速率常数分别比k（cat）大21倍和7倍，表明黄嘌呤对酶的还原半反应在黄嘌呤/NAD周转中主要是限速的。讨论了XDH的稳态机制。
The reaction between reduced xanthine dehydrogenase (XDH) from bovine milk and NAD has been studied in detail. An understanding of this reaction is necessary for a complete description of XDH turnover with its presumed natural electron acceptor and to address the preference of WH for NAD over oxygen as a substrate. The reaction between pre-reduced XDH and NAD was studied by stopped-flow spectrophotometry. The reaction was found to involve two rounds of oxidation with 2 eq of NAD. The first round goes to completion, and the second round reaches a slightly disfavored equilibrium. Rapid binding of NAD with an apparent K-d of 25 +/- 2 mu M is followed by NAD reduction at a rate constant of 130 +/- 13 s(-1). NADH dissociation at a rate constant of 42 +/- 12 s(-1) completes a round of oxidation. These steps have been successfully tested and modeled to repeat themselves in the second round of oxidation. The association rate constant for NAD binding was estimated to be much greater than any rate constant measured in the oxidation by molecular oxygen, thus explaining how NAD competes with oxygen for reducing equivalents. Rate constants for NAD reduction and NADH dissociation are respectively 21- and 7-fold greater than k(cat), indicating that the reductive half-reaction of the enzyme by xanthine is mostly rate-limiting in xanthine/NAD turnover. A steady-state mechanism for XDH is discussed.