Brønsted Analysis and Rate-Limiting Steps for the T5 Flap Endonuclease Catalyzed Hydrolysis of Exonucleolytic Substrates

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• 作者：Blanka Sengerov ; Christopher Tomlinson ; John M. Atack ; Ryan Williams ; Jon R. Sayers ; Nicholas H. Williams ; Jane A. Grasby
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：September 21, 2010
• 年：2010
• 卷：49
• 期：37
• 页码：8085-8093
• 全文大小：929K
• 年卷期：v.49,no.37(September 21, 2010)
• ISSN：1520-4995

文摘

During replication and repair flap endonucleases (FENs) catalyze endonucleolytic and exonucleolytic (EXO) DNA hydrolyses. Altering the leaving group pK_a, by replacing the departing nucleoside with analogues, had minimal effect on k_cat/K_M in a T5FEN-catalyzed EXO reaction, producing a very low Brønsted coefficient, β_lg. Investigation of the viscosity dependence of k_cat/K_M revealed that reactions of EXO substrates are rate limited by diffusional encounter of enzyme and substrate, explaining the small β_lg. However, the maximal single turnover rate of the FEN EXO reaction also yields a near zero β_lg. A low β_lg was also observed when evaluating k_cat/K_M for D201I/D204S FEN-catalyzed reactions, even though these reactions were not affected by added viscogen. But an active site K83A mutant produced a β_lg = −1.2 ± 0.10, closer to the value observed for solution hydrolysis of phosphate diesters. The pH−maximal rate profiles of the WT and K83A FEN reactions both reach a maximum at high pH and do not support an explanation of the data that involves catalysis of leaving group departure by Lys 83 functioning as a general acid. Instead, a rate-limiting physical step, such as substrate unpairing or helical arch ordering, that occurs after substrate association must kinetically hide an inherent large β_lg. It is suggested that K83 acts as an electrostatic catalyst that stabilizes the transition state for phosphate diester hydrolysis. When K83 is removed from the active site, chemistry becomes rate limiting and the leaving group sensitivity of the FEN-catalyzed reaction is revealed.