Kinetics of transhydrogenase reaction catalyzed by the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) imply more than one catalytic nucleotide-binding sites.
Kinetics of transhydrogenase reaction catalyzed by the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) imply more than one catalytic nucleotide-binding sites.
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由线粒体 NADH-泛醌氧化还原酶（复合物 I）催化的转氢酶反应动力学意味着存在多个催化核苷酸结合位点。
DOI：
10.1016/s0014-5793(99)00062-9
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发表时间：
1999
期刊：
影响因子：
3.5
通讯作者：
Vinogradov,AD
中科院分区：
文献类型：
--
作者：
Zakharova,NV;Zharova,TV;Vinogradov,AD
研究了牛心亚线粒体颗粒(SMP)、纯化的复合体I和可溶性三亚单位NADH脱氢酶(FP)催化转氢酶反应(NADH、DD转氢酶还原乙酰吡啶腺嘌呤二核苷酸(APAD+))的稳态动力学。在不运行质子泵转氢酶(EC 1.6.1.1)的条件下，SMP和络合物I的DD转氢酶活性呈现复杂的动力学模式：当底物浓度在较大范围内变化时，速度的双倒数曲线不是线性的。没有二元复合体(乒乓球)机制(对于单一底物结合部位酶的预期)在可变底物的任何范围内工作。在转氢酶反应中，与NADH(Ki=40μM)竞争比与APAD+(Ki=150μM)竞争更有效。FMN氧化还原循环依赖的FP催化的DD转氢酶反应是通过三元络合反应进行的。结果表明，络合物I及其衍生的最简单的催化活性片段(FP)在转氢酶反应中具有多个核苷酸结合位点。
The steady-state kinetics of the transhydrogenase reaction (the reduction of acetylpyridine adenine dinucleotide (APAD+) by NADH, DD transhydrogenase) catalyzed by bovine heart submitochondrial particles (SMP), purified Complex I, and by the soluble three-subunit NADH dehydrogenase (FP) were studied to assess a number of the Complex I-associated nucleotide-binding sites. Under the conditions where the proton-pumping transhydrogenase (EC 1.6.1.1) was not operating, the DD transhydrogenase activities of SMP and Complex I exhibited complex kinetic pattern: the double reciprocal plots of the velocities were not linear when the substrate concentrations were varied in a wide range. No binary complex (ping-pong) mechanism (as expected for a single substrate-binding site enzyme) was operating within any range of the variable substrates. ADP-ribose, a competitive inhibitor of NADH oxidase, was shown to compete more effectively with NADH (Ki=40 μM) than with APAD+(Ki=150 μM) in the transhydrogenase reaction. FMN redox cycling-dependent, FP catalyzed DD transhydrogenase reaction was shown to proceed through a ternary complex mechanism. The results suggest that Complex I and the simplest catalytically competent fragment derived therefrom (FP) possess more than one nucleotide-binding sites operating in the transhydrogenase reaction.