RESUMO
Previous osmotic pressure studies of two nonhomologous dihydrofolate reductase (DHFR) enzymes found tighter binding of the nicotinamide adenine dinucleotide phosphate cofactor upon addition of neutral osmolytes. This result is consistent with water release accompanying binding. In contrast, osmotic stress studies found weaker binding of the dihydrofolate (DHF) substrate for both type I and type II DHFRs in the presence of osmolytes; this observation can be explained if dihydrofolate interacts with osmolytes and shifts the equilibrium from the enzyme-bound state toward the unbound substrate. Nuclear magnetic resonance experiments support this hypothesis, finding that osmolytes interact with dihydrofolate. To consider binding without added osmolytes, a high-pressure approach was used. In this study, the type II enzyme, R67 DHFR, was subjected to high hydrostatic pressure (HHP). Both enzyme activity and fluorescence measurements find the protein tolerates pressures up to 200 MPa. Binding of the cofactor to R67 DHFR weakens with increasing pressure, and a positive association volume of 11.4 ± 0.5 cm(3)/mol was measured. Additionally, an activation volume of 3.3 ± 0.5 cm(3)/mol describing k(cat)/K(m(DHF)) was determined from progress curve analysis. Results from these HHP experiments suggest water release accompanies binding of both the cofactor and DHF to R67 DHFR. In an additional set of experiments, isothermal titration calorimetry studies in H2O and D2O find that water reorganization dominates the enthalpy associated with binding of DHF to R67 DHFR·NADP(+), while no obvious effects occur for cofactor binding. The combined results indicate that water plays an active role in ligand binding to R67 DHFR.
