Conformational substates modulate hydride transfer in dihydrofolate reductase.
In earlier studies of the hydride-transfer reaction catalyzed by dihydrofolate reductase (DHFR) we identified features of the protein correlated with variations in the reaction barrier. We extend the scope of those studies by carrying out potential of mean force (PMF) simulations to determine the hydride-transfer barrier in the wild-type protein as well as the G121V and G121S mutants. While our prior studies focused on the reactant state, our current work addresses the full reaction pathway and directly probes the reactive event. The free energy barriers and structural ensembles resulting from these PMF calculations exhibit the same trends reported in our previous work. Fluctuations present in these simulations also exhibit trends associated with differences in the hydride-transfer barrier height. Moreover, vibrational modes anticipated to promote hydride transfer exhibit larger amplitudes in simulations that generate lowered barriers. The results of our study indicate that discrete basins (substates) on a potential energy landscape of the enzyme give rise to distinct hydride-transfer barriers. We suggest that the long-range effects of mutations at position 121 within DHFR are mediated by differentially preorganized protein environments in the context of distinct substate distributions, with concomitant changes to the dynamic properties of the enzyme.
Thorpe
Ian F
IF
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Brooks
Charles L
CL
D002384:Catalysis; D006859:Hydrogen / Q000737:chemistry*; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*
D006859:Hydrogen; D013762:Tetrahydrofolate Dehydrogenase