Transition State in DNA Polymerase 尾 Catalysis: Rate-Limiting Chemistry Altered by Base-Pair Configuration
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- 作者：Keriann Oertell ; Brian T. Chamberlain ; Yue Wu ; Elena Ferri ; Boris A. Kashemirov ; William A. Beard ; Samuel H. Wilson ; Charles E. McKenna ; Myron F. Goodman
- 刊名：Biochemistry
- 出版年：2014
- 出版时间：March 25, 2014
- 年：2014
- 卷：53
- 期：11
- 页码：1842-1848
- 全文大小：287K
- 年卷期：v.53,no.11(March 25, 2014)
- ISSN：1520-4995
文摘
Kinetics studies of dNTP analogues having pyrophosphate-mimicking 尾,纬-pCXYp leaving groups with variable X and Y substitution reveal striking differences in the chemical transition-state energy for DNA polymerase 尾 that depend on all aspects of base-pairing configurations, including whether the incoming dNTP is a purine or pyrimidine and if base-pairings are right (T鈥 and G鈥) or wrong (T鈥 and G鈥). Br酶nsted plots of the catalytic rate constant (log(kpol)) versus pKa4 for the leaving group exhibit linear free energy relationships (LFERs) with negative slopes ranging from 鈭?.6 to 鈭?.0, consistent with chemical rate-determining transition-states in which the active-site adjusts to charge-stabilization demand during chemistry depending on base-pair configuration. The Br酶nsted slopes as well as the intercepts differ dramatically and provide the first direct evidence that dNTP base recognition by the enzyme鈥損rimer鈥搕emplate complex triggers a conformational change in the catalytic region of the active-site that significantly modifies the rate-determining chemical step.