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

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.