Mycobacterium tuberculosis mycothione reductase:: pH dependence of the kinetic parameters and kinetic isotope effects
Mycobacterium tuberculosis mycothione reductase:: pH dependence of the kinetic parameters and kinetic isotope effects
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DOI：
10.1021/bi0029144
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发表时间：
2001-05-01
期刊：
影响因子：
2.9
通讯作者：
Blanchard, JS
中科院分区：
文献类型：
--
作者：
Patel, MP;Blanchard, JS
最近在结核分枝杆菌中鉴定了一种酶，它能催化nadph依赖性的独特的低还原；分子量二硫化菌硫酮，菌硫酮还原酶，使我们更详细地研究了催化机制。动力学参数V和V/K对NADPH、NADH和二硫化菌硫酮活性类似物的pH依赖性。已测定了肌醇菌硫酮二硫化。通过对FH谱的分析，初步确定了对二硫还原机制至关重要的催化残基，即His444-Glu449离子对和Cys39。溶剂动力学同位素对V和V/K-DIMSSM的产率分别为1.7 +/- 0.2和1.4 +/- 0.2，但对V/K-NADPH没有影响。质子库存研究；（V与D2O摩尔分数）呈线性关系，表明在溶剂同位素敏感步骤中发生了单质子转移。用NADPH和NADH测定了稳态氘动力学同位素对V的影响，产值分别为1.27 +/- 0.03和1.66 +/- 0.14。稳态前初生氘对NADPH和NADH酶还原的动力学同位素效应值分别为1.82 +/- 0.04和1.59 +/- 0.06。利用NADH得到的稳态原生氘动力学同位素效应与单次翻转条件下得到的结果一致，表明本征原生动力学同位素效应得到了完整的表达。利用NADPH和NADH对还原半反应进行快速反应研究，最大反应速率分别为129 +/- 2和20 +/- 1 s(-1)，而用真菌硫醇二硫化物氧化双电子还原酶的类似研究，最大反应速率为190 +/- 10 s（-1）。这些数据表明了一种独特的黄蛋白二硫机制，其中氧化半反应的速率略快于还原半反应的速率。
The recent identification of the enzyme in Mycobacterium tuberculosis that catalyzes the NADPH-dependent reduction of the unique low; molecular weight disulfide mycothione, mycothione reductase, has led us to examine the mechanism of catalysis in greater detail. The pH dependence of the kinetic parameters V and V/K for NADPH, NADH, and an active analogue of mycothione disulfide. desmyo-inositol mycothione disulfide, has been determined. An analysis of the FH profiles has allowed the tentative assignment of catalytically significant residues crucial to the mechanism of disulfide reduction, namely, the His444-Glu449 ion pair and Cys39. Solvent kinetic isotope effects were observed on V and V/K-DIMSSM, yielding values of 1.7 +/- 0.2 and 1.4 +/- 0.2, respectively, but not on V/K-NADPH. Proton inventory studies; (V versus mole fraction of D2O) were linear, indicative of a single proton transfer in a solvent isotopically sensitive step. Steady-state primary deuterium kinetic isotope effects on V have been determined using NADPH and NADH, yielding values of 1.27 +/- 0.03 and 1.66 +/- 0.14, respectively. The pre-steady state primary deuterium kinetic isotope effect on enzyme reduction has values of 1.82 +/- 0.04 and 1.59 +/- 0.06 for NADPH and NADH, respectively. The steady-state primary deuterium kinetic isotope effect using NADH coincide with that obtained under single turnover conditions, suggesting the complete expression of the intrinsic primary kinetic isotope effect. Rapid reaction studies on the reductive half-reaction using NADPH and NADH yielded maximal rates of 129 +/- 2 and 20 +/- 1 s(-1), respectively, while similar studies of the oxidation of the two-electron reduced enzyme by mycothiol disulfide yielded a maximum rate of 190 +/- 10 s(-1). These data suggest a unique flavoprotein disulfide mechanism in which the rate of the oxidative half-reaction is slightly faster than the rate of the reductive half-reaction.