Role of Active Site Loop Dynamics in Mediating Ligand Release from Dihydrofolate Reductase

Conformational fluctuations from ground-state to sparsely populated but functionally important excited states play a key role in enzyme catalysis. For 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 DHFR, where the sequence in the Met20 loop is replaced by the human sequence (N23PP/S148A), models human DHFR and is incapable of accessing the occluded conformation. H and N 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.