Kinetic Mechanism of Active Site Assembly and Chemical Catalysis of DNA Polymerase 尾

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• 作者：Paul B. Balbo ; Eric Chun-Wei Wang ; Ming-Daw Tsai
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：November 15, 2011
• 年：2011
• 卷：50
• 期：45
• 页码：9865-9875
• 全文大小：527K
• 年卷期：v.50,no.45(November 15, 2011)
• ISSN：1520-4995

文摘

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 Mg²⁺-coordinating ligands, and proton transfer. Here we address this broad issue by conducting extensive transient state kinetic analyses of DNA polymerase 尾 (Pol 尾). 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鈥?O^{鈥?/sup> 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 [Mg2+] 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 尾 and also reconcile some computational and experimental findings.}