Abstract
Conformational fluctuations from ground-state to sparsely populated but functionally important excited states play a key role in enzyme catalysis. For Escherichia coli dihydrofolate reductase (DHFR), the release of the product tetrahydrofolate (THF) and oxidized cofactor NADP+ occurs through exchange between closed and occluded conformations of the Met20 loop. A "dynamic knockout" mutant of E. coli DHFR, where the E. coli sequence in the Met20 loop is replaced by the human sequence (N23PP/S148A), models human DHFR and is incapable of accessing the occluded conformation. 1H and 15N CPMG relaxation dispersion analysis for the ternary product complex of the mutant enzyme with NADP+ and the product analogue 5,10-dideazatetrahydrofolate (ddTHF) (E:ddTHF:NADP+) reveals the mechanism by which NADP+ is released when the Met20 loop cannot undergo the closed-to-occluded conformational transition. Two excited states were observed: one related to a faster, relatively high-amplitude conformational fluctuation in areas near the active site, associated with the shuttling of the nicotinamide ring of the cofactor out of the active site, and the other to a slower process where ddTHF undergoes small-amplitude motions within the binding site that are consistent with disorder observed in a room-temperature X-ray crystal structure of the N23PP/S148A mutant protein. These motions likely arise due to steric conflict of the pterin ring of ddTHF with the ribose-nicotinamide moiety of NADP+ in the closed active site. These studies demonstrate that site-specific kinetic information from relaxation dispersion experiments can provide intimate details of the changes in catalytic mechanism that result from small changes in local amino acid sequence.