The role of the putative catalytic base in the phosphoryl transfer reaction in a protein kinase: First-principles calculations
The role of the putative catalytic base in the phosphoryl transfer reaction in a protein kinase: First-principles calculations
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DOI：
10.1021/ja029618u
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发表时间：
2003-08-20
影响因子：
15
通讯作者：
Weare, JH
中科院分区：
文献类型：
--
作者：
Valiev, M;Kawai, R;Weare, JH
蛋白激酶是通过ATP的γ-磷酸转移到靶蛋白来控制大多数细胞信号事件的重要酶。即使经过多年的研究，这种反应的机制仍然知之甚少。在许多可能导致这种酶的1011倍速率提高的因素中，保守的天冬氨酸（Asp166）的作用被给予了特别的考虑。虽然突变研究已经确定了Asp166的基本存在，但其功能仍存在争议。由于反应速率的pH依赖性、同位素测量和预稳态动力学，基于活性位点位置分配给Asp166的一般碱催化剂作用受到了质疑。最近的半经验计算增加了围绕Asp166在催化机制中的作用的争议。在这些计算中没有发现Asp166的主要作用，这些计算预测了反应过程，包括底物质子早期转移到磷酸基上。这些结论与实验观察结果不一致。为了用更可靠的计算方法解决实验结果与理论之间的这些差异，并为理解这一重要酶家族的催化作用提供理论平台，我们对cAPK激酶的反应过程进行了第一性原理结构和动力学计算。为了保留反应的基本特征，计算中包含了所有关键保守残基（82个原子）的表示。采用混合B3LYP和PBE96广义梯度近似的局部基密度泛函（DFT）方法进行结构计算。这种计算已被证明可以为大量系统提供高度精确的结构信息。优化后的反应物态结构与x射线数据吻合较好。与半经验方法相比，最低能积态将底物质子置于Asp166上。第一性原理分子动力学模拟为该产物状态的稳定性提供了额外的支持。后者还表明，质子转移到Asp166发生在反应中PO桥接位置的键裂解已经进行的点上。这一机制进一步得到了过渡态计算结构的支持，在过渡态中，底物羟基基本上是完整的。在过渡态中存在一种类似于偏磷酸酯的结构，这与过渡态模拟物的x射线结构一致。根据计算的过渡态结构，估计其解离率为85%。我们的分析还表明，Asp166与底物羟基之间的氢键强度增加，而后者在过渡状态下的键强度略有下降。总之，我们的计算证明了Asp166作为质子受体在酶促机制中的重要性。然而，从底物中提取质子发生在反应过程的后期。因此，在cAPK蛋白激酶的催化机制中，Asp166扮演了一个“质子陷阱”的角色，将转移的磷酸化基锁定在底物上。这些结果解决了先前理论与实验之间的矛盾，并对Asp166在蛋白激酶催化机制中的作用有了新的认识。
Protein kinases are important enzymes controlling the majority of cellular signaling events via a transfer of the γ-phosphate of ATP to a target protein. Even after many years of study, the mechanism of this reaction is still poorly understood. Among many factors that may be responsible for the 1011-fold rate enhancement due to this enzyme, the role of the conserved aspartate (Asp166) has been given special consideration. While the essential presence of Asp166 has been established by mutational studies, its function is still debated. The general base catalyst role assigned to Asp166 on the basis of its position in the active site has been brought into question by the pH dependence of the reaction rate, isotope measurements, and pre-steady-state kinetics. Recent semiempirical calculations have added to the controversy surrounding the role of Asp166 in the catalytic mechanism. No major role for Asp166 has been found in these calculations, which have predicted the reaction process consisting of an early transfer of a substrate proton onto the phosphate group. These conclusions were inconsistent with experimental observations. To address these differences between experimental results and theory with a more reliable computational approach and to provide a theoretical platform for understanding catalysis in this important enzyme family, we have carried out first-principles structural and dynamical calculations of the reaction process in cAPK kinase. To preserve the essential features of the reaction, representations of all of the key conserved residues (82 atoms) were included in the calculation. The structural calculations were performed using the local basis density functional (DFT) approach with both hybrid B3LYP and PBE96 generalized gradient approximations. This kind of calculation has been shown to yield highly accurate structural information for a large number of systems. The optimized reactant state structure is in good agreement with X-ray data. In contrast to semiempirical methods, the lowest energy product state places the substrate proton on Asp166. First-principles molecular dynamics simulations provide additional support for the stability of this product state. The latter also demonstrate that the proton transfer to Asp166 occurs at a point in the reaction where bond cleavage at the PO bridging position is already advanced. This mechanism is further supported by the calculated structure of the transition state in which the substrate hydroxyl group is largely intact. A metaphoshate-like structure is present in the transition state, which is consistent with the X-ray structures of transition state mimics. On the basis of the calculated structure of the transition state, it is estimated to be 85% dissociative. Our analysis also indicates an increase in the hydrogen bond strength between Asp166 and substrate hydroxyl and a small decrease in the bond strength of the latter in the transition state. In summary, our calculations demonstrate the importance of Asp166 in the enzymatic mechanism as a proton acceptor. However, the proton abstraction from the substrate occurs late in the reaction process. Thus, in the catalytic mechanism of cAPK protein kinase, Asp166 plays a role of a “proton trap” that locks the transferred phosphoryl group to the substrate. These results resolve prior inconsistencies between theory and experiment and bring new understanding of the role of Asp166 in the protein kinase catalytic mechanism.