Millisecond Dynamics in Glutaredoxin during Catalytic Turnover Is Dependent on Substrate Binding and Absent in the Resting States
Millisecond Dynamics in Glutaredoxin during Catalytic Turnover Is Dependent on Substrate Binding and Absent in the Resting States
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DOI：
10.1021/ja1096539
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发表时间：
2011-03-09
影响因子：
15
通讯作者：
Teilum, Kaare
中科院分区：
文献类型：
--
作者：
Jensen, Kristine Steen;Winther, Jakob R.;Teilum, Kaare
构象动力学对酶的功能至关重要。然而，哪些酶的运动决定了催化效率，以及相同的运动是否对所有酶都很重要，目前还不清楚。在这里，我们通过核磁共振磁化转移、R-2弛豫分散和配体滴定实验，研究了催化周转过程中戊二醛的构象动力学。谷胱甘肽催化谷胱甘肽交换反应，形成稳定的谷胱甘肽化酶中间体。还原态和谷胱甘肽化态之间的平衡是在毫秒时间尺度上进行生物化学调整的。用N-15核自旋弛豫色散实验跟踪了催化过程中蛋白质骨架的构象变化。23个残基的构象转变可以很好地描述为两态过程，其交换速率与谷胱甘肽交换速率相对应。还原谷胱甘肽的结合导致了还原酶的竞争性抑制，其动力学与反应相似。这一观察结果将催化过程中观察到的运动直接耦合到底物结合上。在静息状态下，没有观察到酶在催化周转的时间尺度上的骨架运动，这意味着替代构象不会积累到显著的浓度。这些结果表明，谷氨酰胺还蛋白的周转率受生产酶底物相遇复合物的形成控制，催化作用是通过诱导拟合机制进行的，而不是由内在构象动力学驱动的构象选择。
Conformational dynamics is important for enzyme function. Which motions of enzymes determine catalytic efficiency and whether the same motions are important for all enzymes, however, are not well understood. Here we address conformational dynamics in glutaredoxin during catalytic turnover with a combination of NMR magnetization transfer, R-2 relaxation dispersion, and ligand titration experiments. Glutaredoxins catalyze a glutathione exchange reaction, forming a stable glutathinoylated enzyme intermediate. The equilibrium between the reduced state and the glutathionylated state was biochemically tuned to exchange on the millisecond time scale. The conformational changes of the protein backbone during catalysis were followed by N-15 nuclear spin relaxation dispersion experiments. A conformational transition that is well described by a two-state process with an exchange rate corresponding to the glutathione exchange rate was observed for 23 residues. Binding of reduced glutathione resulted in competitive inhibition of the reduced enzyme having kinetics similar to that of the reaction. This observation couples the motions observed during catalysis directly to substrate binding. Backbone motions on the time scale of catalytic turnover were not observed for the enzyme in the resting states, implying that alternative conformers do not accumulate to significant concentrations. These results infer that the turnover rate in glutaredoxin is governed by formation of a productive enzyme substrate encounter complex, and that catalysis proceeds by an induced fit mechanism rather than by conformer selection driven by intrinsic conformational dynamics.