Date of Award

Winter 2009

Document Type


Degree Name

Doctor of Philosophy (PhD)

Committee Director

Jennifer E. Poutsma

Committee Member

Kenneth G. Brown

Committee Member

Patricia A. Pleban

Committee Member

Colm Whelan

Committee Member

X. Nancy Xu


R67 dihydrofolate reductase (R67 DHFR) is a plasmid encoded enzyme which catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as a cofactor. R67 DHFR is a homo-tetramer and D2 symmetric. It contains only one active site, which spans the central channel of the enzyme. The active site can bind either two reactants (DHF), two cofactors (NADPH) or one of each (NADPH/DHF), which is the productive ternary complex (i.e. the complex which yields product). In order to favor formation of the productive complex, this enzyme exhibits binding cooperativity. Unlike other allosteric enzymes which achieve binding cooperativity through conformational changes, this enzyme does not appear to undergo any backbone alterations upon ligand binding. Therefore, a different mechanism must be involved in the cooperativity. In this study, computational approaches were employed to better understand the source of the unique binding patterns of this enzyme.

Molecular dynamics simulations were initially performed on complexes involving truncated ligands and then the full ligands and mutants were studied. Agreement was found between the simulated results and experimental data. A well-maintained backbone conformation was observed for all simulated complexes and the calculated binding energies were able to reproduce the cooperative binding patterns for the truncated and full ligand studies. Residue Q67 can fold down to create additional room for the ligands in the active site while Y69 can fortify the H-bonding network to NADPH. The largest contribution to the binding cooperativity came from ligand-ligand interactions and electrostatic interactions. The DHF tail is extremely flexible and accounts for the lower binding energy of DHF. In addition, the ring area of DHF was also found to be flexible and adopted different binding positions. Losing the interaction to residue K32 significantly reduced DHF's binding energy while this effect is less profound for NADPH. Simulations on the mutants suggest that the NADPH binding conformation did not undergo any significant changes. The correct binding site for a single DHF ligand remains unclear as the results were unable to reproduce the tighter binding to the mutant.