The reaction of reduced xanthine dehydrogenase with molecular oxygen - Reaction kinetics and measurement of superoxide radical
The reaction of reduced xanthine dehydrogenase with molecular oxygen - Reaction kinetics and measurement of superoxide radical
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DOI：
10.1074/jbc.272.13.8370
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发表时间：
1997-03-28
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
牛乳中的黄嘌呤脱氢酶（XDH）在黄嘌呤/氧周转试验中具有显著的活性。本文详细研究了XDH在25 ℃、pH7.5条件下与分子氧的氧化半反应，以确定XDH优先选择NAD而不是氧作为氧化底物的依据。采用停流分光光度法跟踪XDH氧化过程中的光谱变化，考察氧化过程中超氧阴离子自由基的生成量，评价XDH催化氧自由基生成的能力。还原的XDH在至少4个双分子步骤中与氧反应，其中每摩尔XDH由最后2个电子氧化形成1.7-1.9摩尔超氧化物。讨论了一个模型，其中黄素氢醌以至少72的速率常数将电子转移到氧以产生过氧化氢，XDH的黄嘌呤/氧和NADH/氧转换的稳态动力学被确定为h（cat）值为2.1 +/-。0.1和2.5 +/-0.9s（-1），因此XDH能够以黄嘌呤/NAD转换速率6.3s（-1）的三分之一催化还原氧物质的形成（Hunt，J.，和Massey，V.（1992）J.Biol.Chem.267，21479-21485）。由于XDH含有显著的和内在的黄嘌呤氧化酶活性，因此必须谨慎地仅基于转换测定来估计XO和XDH的相对量。含有不同量的黄嘌呤、NAD和氧的初始速率测定表明，在100%氧饱和度下，对于每种底物，仅在黄嘌呤和NAD浓度低于K-m时才抑制NADH形成。
Xanthine dehydrogenase (XDH) from bovine milk contains significant activity in xanthine/oxygen turnover assays, The oxidative half-reaction of XDH with molecular oxygen has been studied in detail, at 25 degrees C, pH 7.5, to determine the basis of the preference of XDH for NAD over oxygen as oxidizing substrate. Spectral changes of XDH accompanying oxidation were followed by stopped-flow spectrophotometry, The amount of superoxide radicals formed during oxidation was investigated to assess the ability of XDH to catalyze production of oxygen radicals. Reduced XDH reacts with oxygen in at least 4 bi-molecular steps, with 1.7-1.9 mol of superoxide per mol of XDH formed from the last 2 electrons oxidized. A model is discussed in which the flavin hydroquinone transfers electrons to oxygen to produce hydrogen peroxide at a rate constant of at least 72,000 M(-1) s(-1) whereas flavin semiquinone reduces oxygen to form superoxide as slow as 16 M(-1) s(-1).Steady-state kinetics of xanthine/oxygen and NADH/oxygen turnover of XDH were determined to have h(cat) values of 2.1 +/- 0.1 and 2.5 +/- 0.9 s(-1), respectively, at 25 degrees C, pH 7.5, XDH is therefore capable of catalyzing the formation of reduced oxygen species at one-third the rate of xanthine/NAD turnover, 6.3 s(-1) (Hunt, J., and Massey, V. (1992) J. Biol. Chem. 267, 21479-21485), in the absence of NAD. As XDH contains a significant and intrinsic xanthine oxidase activity, estimates of relative amounts of XO and XDH based solely upon turnover assays must be made with caution, Initial-rate assays containing varying amounts of xanthine, NAD, and oxygen indicate that at 100% oxygen saturation, NADH formation is only inhibited at concentrations of xanthine and NAD below K-m for each substrate.