Institute of Nanobiology and Structural Biology, Global Change Research Center, Academy of Sciences of Czech Republic, Nove Hrady, Czech Republic.
PLoS One. 2012;7(8):e43902. doi: 10.1371/journal.pone.0043902. Epub 2012 Aug 29.
The E. coli protein WrbA is an FMN-dependent NAD(P)H:quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive patterns of product inhibition, though consistent with the general class of bi-substrate reactions, do not exclude a minor contribution from additional forms of the enzyme. NMR results support the presence of additional enzyme forms. Docking and energy calculations find that electron-transfer-competent binding sites for NADH and benzoquinone present severe steric overlap, consistent with the ping-pong mechanism. Unexpectedly, plots of initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depending on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process. WrbA shares these and other details of its kinetic behavior with mammalian DT-diaphorase, an FAD-dependent NAD(P)H:quinone oxidoreductase. An extensive literature review reveals several other enzymes with two-plateau kinetic plots, but in no case has a molecular explanation been elucidated. Preliminary sedimentation velocity analysis of WrbA indicates a large shift in size of the multimer with temperature, suggesting that subunit assembly coupled to substrate binding may underlie the two-plateau behavior. An additional aim of this report is to bring under wider attention the apparently widespread phenomenon of two-plateau Michaelis-Menten plots.
大肠杆菌蛋白 WrbA 是一种依赖 FMN 的 NAD(P)H：醌氧化还原酶，与氧化防御有关。四聚体酶的三个亚基分别贡献于四个相同的、洞穴状的活性位点，这些活性位点似乎可以容纳 NAD(P)H 或各种醌，但不能同时容纳，这表明存在一种必需的四聚体和乒乓机制，其中 NAD 在氧化的醌结合之前离开。本研究旨在评估这些建议，并表征 WrbA 的动力学行为。稳态动力学结果表明，WrbA 符合乒乓机制，表现在随着底物浓度的变化，表观 Vmax 与 Km 比值保持不变。然而，产物抑制的竞争性/非竞争性模式虽然与双底物反应的一般类别一致，但不能排除酶的其他形式的少量贡献。NMR 结果支持存在额外的酶形式。对接和能量计算发现，NADH 和苯醌的电子转移有效结合位点存在严重的空间重叠，与乒乓机制一致。出乎意料的是，根据酶在测定前保持的温度，初始速度与 NADH 或苯醌浓度的关系图呈现出一个或两个米氏-门坦阶段。温度的影响是可逆的，这表明存在分子内构象过程。WrbA 与其动力学行为相似的哺乳动物 DT-二氢二醇脱氢酶（一种依赖 FAD 的 NAD(P)H：醌氧化还原酶）。广泛的文献回顾揭示了其他几种具有两平台动力学图的酶，但在任何情况下都没有阐明分子解释。WrbA 的初步沉降速度分析表明，随着温度的变化，多聚体的大小发生了很大的变化，这表明亚基组装与底物结合可能是两平台行为的基础。本报告的另一个目的是引起更广泛的关注，即显然广泛存在的两平台米氏-门坦图现象。
