Zakharova N V, Zharova T V, Vinogradov A D
Department of Biochemistry, School of Biology, Moscow State University, Russian Federation.
FEBS Lett. 1999 Feb 12;444(2-3):211-6. doi: 10.1016/s0014-5793(99)00062-9.
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 microM) than with APAD+ (Ki = 150 microM) 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.
研究了牛心亚线粒体颗粒（SMP）、纯化的复合体I以及可溶性三聚体NADH脱氢酶（FP）催化的转氢酶反应（NADH还原乙酰吡啶腺嘌呤二核苷酸（APAD+），即DD转氢酶）的稳态动力学，以评估一些与复合体I相关的核苷酸结合位点。在质子泵转运转氢酶（EC 1.6.1.1）不发挥作用的条件下，SMP和复合体I的DD转氢酶活性呈现出复杂的动力学模式：当底物浓度在很宽的范围内变化时，速度的双倒数图不是线性的。在可变底物的任何范围内，均不存在二元复合物（乒乓）机制（这是单底物结合位点酶所预期的）。ADP-核糖是NADH氧化酶的竞争性抑制剂，在转氢酶反应中，它与NADH（Ki = 40 microM）的竞争比与APAD+（Ki = 150 microM）更有效。由FMN氧化还原循环依赖性的、FP催化的DD转氢酶反应显示通过三元复合物机制进行。结果表明，复合体I及其衍生的最简单的具有催化活性的片段（FP）在转氢酶反应中具有不止一个核苷酸结合位点。
