A Stopped Flow Transient Kinetic Analysis of Substrate Binding and Catalysis in Escherichia coli D-3-Phosphoglycerate Dehydrogenase
A Stopped Flow Transient Kinetic Analysis of Substrate Binding and Catalysis in Escherichia coli D-3-Phosphoglycerate Dehydrogenase
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DOI：
10.1074/jbc.m805180200
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发表时间：
2008-10-31
影响因子：
4.8
通讯作者：
Grant, Gregory A.
中科院分区：
文献类型：
--
作者：
Burton, Rodney L.;Hanes, Jeremiah W.;Grant, Gregory A.
通过跟踪蛋白质色氨酸的荧光和从蛋白质色氨酸到结合的NADH的荧光共振能量转移来进行大肠杆菌D-3-磷酸甘油酸脱氢酶的预稳态、停流分析。结果表明，底物的结合是有序的，辅酶，NADH，首先结合。此外，分析表明，有两套网站上的四聚体酶，可以区分他们的动力学行为。NADH结合是一致的初始结合事件，然后由一个缓慢的构象变化为每个网站。这种缓慢的构象变化是NADH与脱辅基酶表观紧密结合的原因，但当酶快速翻转时，这种构象变化太慢而不能参与催化循环。随后结合的底物，α-酮戊二酸，其特征在于一个快速的平衡结合事件，然后由每个网站的构象变化。NAD（+）还原方向的催化显示出明显的活性爆发，随后是缓慢的周转率，表明限速步骤是在氢化物转移之后。在NADH氧化的方向上的催化没有显示突发动力学，表明限速步骤是在或之前的氢化物转移步骤。爆发数据表明，NAD（+）还原速率（3.8 s（-1））与该方向上酶的k（cat）（2-3 s（-1））相似。然而，对与氘代NADH的反应的分析未能显示对反应速度的影响，aV（H）/V-D = 1.07 +/- 0.06。通过停流分析确定的其他速率都不能解释酶在任一方向上的k（cat）（正向k（cat）= 0.01 s（-1），反向k（cat）= 2-3 s（-1）），这表明两个方向上的速率限制步骤是酶的构象变化，而这种变化是光学检测不到的。
Pre-steady state, stopped flow analysis of Escherichia coli D-3-phosphoglycerate dehydrogenase was performed by following the fluorescence of protein tryptophan and the fluorescence resonance energy transfer from protein tryptophan to bound NADH. The results indicate that binding of substrates is ordered, with coenzyme, NADH, binding first. Furthermore, the analysis indicated that there are two sets of sites on the tetrameric enzyme that can be differentiated by their kinetic behavior. NADH binding was consistent with an initial binding event followed by a slow conformational change for each site. The slow conformational change is responsible for the apparent tight binding of NADH to the apoenzyme but is too slow to participate in the catalytic cycle when the enzyme is rapidly turning over. Subsequent binding of the substrate, alpha-ketoglutarate, was characterized by a rapid equilibrium binding event followed by a conformational change for each site. Catalysis in the direction of NAD(+) reduction showed a distinct burst of activity followed by a slow rate of turnover, indicating that the rate-limiting step is after hydride transfer. Catalysis in the direction of NADH oxidation did not display burst kinetics, indicating that the rate-limiting step is at or before the hydride transfer step. The burst data indicated that the rate of NAD(+) reduction (3.8 s(-1)) is similar to the k(cat) of the enzyme (2-3 s(-1)) in that direction. However, analysis of the reaction with deuterated NADH failed to show an effect on the velocity of the reaction with aV(H)/V-D = 1.07 +/- 0.06. None of the other rates determined by stopped flow analysis could account for the k(cat) of the enzyme in either direction (forward k(cat) = 0.01 s(-1), reverse k(cat) = 2-3 s(-1)), suggesting that the rate-limiting step in both directions is a conformational change in the enzyme that is not detected optically.