Institute of Biological Chemistry, Academia Sinica, Nangang, Taipei 115, Taiwan.
Biochemistry. 2011 Nov 15;50(45):9865-75. doi: 10.1021/bi200954r. Epub 2011 Oct 19.
It has been inferred from structural and computational studies that the mechanism of DNA polymerases involves subtle but important discrete steps that occur between binding and recognition of the correct dNTP and chemical catalysis. These steps potentially include local conformational changes involving active site residues, reorganization of Mg(2+)-coordinating ligands, and proton transfer. Here we address this broad issue by conducting extensive transient state kinetic analyses of DNA polymerase β (Pol β). We also performed kinetic simulations to evaluate alternative kinetic models. These studies provide some support for two-step subdomain closing and define constraints under which a kinetically significant prechemistry step can occur. To experimentally identify additional microscopic steps, we developed a stopped flow absorbance assay to measure proton formation that occurs during catalysis. These studies provide direct evidence that formation of the enzyme-bound 3'-O(-) nucleophile is rate determining for chemistry. We additionally show that at low pH the chemical step is rate limiting for catalysis, but at high pH, a postchemistry conformational step is rate limiting due to a pH-dependent increase in the rate of nucleotidyl transfer. Finally, we performed exhaustive analyses of [Mg(2+)] and pH effects. In contrast to published studies, the results suggest an irregular pH dependence of k(pol), which is consistent with general base catalysis involving cooperativity between two or more protonic residues. Overall, the results represent significant advancement in the kinetic mechanism of Pol β and also reconcile some computational and experimental findings.
已经从结构和计算研究推断出，DNA 聚合酶的机制涉及在结合和识别正确的 dNTP 以及化学催化之间发生的微妙但重要的离散步骤。这些步骤可能包括涉及活性位点残基的局部构象变化、Mg（2+）配位配体的重新组织以及质子转移。在这里，我们通过对 DNA 聚合酶 β（Pol β）进行广泛的瞬态动力学分析来解决这个广泛的问题。我们还进行了动力学模拟以评估替代的动力学模型。这些研究为两步亚基关闭提供了一些支持，并定义了可以发生具有动力学意义的预化学步骤的约束条件。为了实验鉴定其他微观步骤，我们开发了一种停流吸收测定法来测量催化过程中发生的质子形成。这些研究提供了直接证据，证明酶结合的 3'-O（-）亲核体的形成对于化学是决定速率的步骤。我们还表明，在低 pH 下，化学步骤是催化的限速步骤，但在高 pH 下，由于质子转移速率随 pH 依赖性增加，后化学构象步骤是限速步骤。最后，我们对 [Mg（2+）]和 pH 效应进行了详尽的分析。与已发表的研究相反，结果表明 k（pol）的 pH 依赖性不规则，这与涉及两个或更多质子残基之间协同作用的通用碱催化一致。总体而言，这些结果代表了 Pol β 动力学机制的重大进展，也调和了一些计算和实验结果。
