Contribution of Water and Energetics of Ligand Binding in the Catalytic Mechanism of R67 Dihydrofolate Reductase

R67 dihydrofolate reductase (DHFR) catalyzes the transfer of a hydride ion from NADPH to dihydrofolate (DHF) to produce tetrahydrofolate (THF). The enzyme is a homotetramer and its 222 symmetry allows for binding of both ligands to a single active site pore. A productive ternary complex is formed by the binding of one molecule of DHF and NADPH and inter-ligand cooperativity has been suggested to be essential for binding and catalysis. To gain further insight into the thermodynamics involved in the ground state and the transition state, temperature dependent studies on DHF binding and catalysis were performed. It was observed that binding of both NADPH and DHF is enthalpy driven. From van’t Hoff plots, the change in enthalpy, entropy and free energy for NADPH binding to R67 DHFR in the ground state were determined. Similarly, the thermodynamics of DHF binding to the R67 DHFR-NADPH complex in the ground state were determined. Arrhenius plots were also employed to study the energetics of the transition state. A comparison of TdeltaS values (for DHF binding to R67 DHFR-NADPH complex) in both ground state and transition state indicates that TdeltaS is more negative in the transition state (–11.3 kcal/mol) as compared to the ground state (–5.4 kcal/mol). This indicates a reorientation of the substrate in the transition state.

The role of water in DHF and NADPH binding to R67 DHFR was also investigated. For this, the effect of osmotic pressure on the K_a /K_m of ligand binding, as well as the k_cat of the reaction was studied. It was observed that the k_cat of the reaction was not significantly affected, while the binding of ligands was affected with increasing osmolality. Specifically, binding of NADPH tightened as osmolality increased, while binding of DHF weakened with increasing osmolality, suggesting release of water upon NADPH binding and an uptake of water on DHF binding. Results from in vivo experiments on E.coli cells containing wild type and mutant clones of R67 DHFR were also consistent with in vitro experiments, suggesting that water is involved in ligand binding to R67 DHFR.