Date of Award
Master of Science
Biochemistry and Cellular and Molecular Biology
Elizabeth E. Howell
Daniel M. Roberts and Engin S Serpersu
Chromosomal dihydrofolate reductase (DHFR) enzymatically reduces dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as a cofactor. R67 DHFR is an R-plasmid encoded enzyme that confers resistance to trimethoprim (TMP), an antibacterial drug. It shares no structural homology with TMP targeted, chromosomal DHFRs.
Previous osmolyte studies in our lab have indicated that DHF binding to R67 DHFR is accompanied by water uptake and NADPH binding is accompanied by water release. These data suggest that water plays a role in balancing the binding affinity. This may happen as R67 DHFR has a generalized binding surface and may need differential water effects to accommodate both ligands. To further examine this hypothesis, we collect binding and steady state kinetic data using hydrostatic pressure. Increasing hydrostatic pressure hydrates molecules and can essentially test the effect of increasing water concentration upon binding. Hydrostatic pressure can also affect the volume of the active site as well.
An activation volume, defined as the change in molar volume associated with the ternary E-NADPH-DHF complex going to the transition state, can be determined from a plot of the natural log of kcat vs pressure. The slope of this line is equal to - Δ[delta]V/RpT. A small slope giving an activation volume of - 1.03 ± 0.9 cm3/mol is observed until 200 Mpa. A second slope describing the effect of pressure from 200 Mpa to 500 Mpa on the activation volume was equal to 8.06 ± 0.8 cm3/mol. Positive activation volumes indicate that the rate-limiting step described accompanies a protein volume increase.
As water reorganization may be playing a role in binding of both substrate and cofactor, studies using isothermal titration calorimetry in both H2O and D2O were utilized to determine the enthalpy of solvent reorganization. The observed enthalpy of the interaction between protein and substrate can be broken up into Δ[delta]Hi (enthalpy of the interaction) and Δ[delta]Hs (enthalpy of solvent reorganization). Since the enthalpy of a hydrogen bond in D2O is approximately 10% greater than in H2O, the ∆[delta]Hs can be estimated.
Timson, Mary Jane, "Getting Heavy: An Exploration into the Effects of D2O and High Hydrostatic Pressure on R67 Dihydrofolate Reductase. " Master's Thesis, University of Tennessee, 2011.