Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers: temporal order and the roles of Asp27 and Tyr100.

ABSTRACT: The reaction catalyzed by Escherichia coli dihydrofolate reductase (ecDHFR) has become a model for understanding enzyme catalysis, and yet several details of its mechanism are still unresolved. Specifically, the mechanism of the chemical step, the hydride transfer reaction, is not fully resolved. We found, unexpectedly, the presence of two reactive ternary complexes [enzyme:NADPH:7,8-dihydrofolate (E:NADPH:DHF)] separated by one ionization event. Furthermore, multiple kinetic isotope effect (KIE) studies revealed a stepwise mechanism in which protonation of the DHF precedes the hydride transfer from the nicotinamide cofactor (NADPH) for both reactive ternary complexes of the WT enzyme. This mechanism was supported by the pH- and temperature-independent intrinsic KIEs for the C-H→C hydride

SUBMITTER: Liu CT 

PROVIDER: S-EPMC4280594 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature