Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University, Bowling Green, Ohio 43403, USA.
J Am Chem Soc. 2011 Sep 14;133(36):14389-95. doi: 10.1021/ja204644y. Epub 2011 Aug 19.
The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. Using single-molecule fluorescence resonance energy transfer (FRET) with a combined statistical data analysis approach, we have identified the intermittently appearing coherence of the enzymatic conformational state from the recorded single-molecule intensity-time trajectories of enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in catalytic reaction. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multistep conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. The coherence frequency, identified by statistical results of the correlation function analysis from single-molecule FRET trajectories, increases with the increasing substrate concentrations. The intermittent coherence in conformational state changes at the enzymatic reaction active site is likely to be common and exist in other conformation regulated enzymatic reactions. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.
蛋白质构象动力学与酶反应之间的关系一直是现代酶学的一个基本焦点。我们使用单分子荧光共振能量转移（FRET）和联合统计数据分析方法，从酶 6-羟甲基-7,8-二氢蝶呤焦磷酸激酶（HPPK）在催化反应中单分子强度-时间轨迹中记录到酶的构象状态间歇性出现相干性。相干构象状态动力学表明，酶催化涉及沿着底物-酶复合物形成和产物释放坐标的多步构象运动，表现出与我们之前报道的时间聚集效应内在相关的极端动态行为。相干频率由单分子 FRET 轨迹的相关函数分析的统计结果确定，随着底物浓度的增加而增加。在酶反应活性位点构象变化中的间歇性相干性可能是普遍存在的，并存在于其他构象调节的酶反应中。我们对 HPPK 与底物相互作用的结果支持了一种多构象态模型，与互补构象选择和诱导契合酶环门构象变化机制一致，在底物-酶活性复合物形成中。
