Identifying the catalytic and ligand binding roles of active site residues in homotetrameric R67 dihydrofolate reductase

by 1970- Strader, Michael Brad

R67 dihydrofolate reductase (DHFR) is a novel protein that confers clinical resistance to trimethoprim (TMP). Surprisingly, this R-plasmid encoded enzyme does not share homology with chromosomal DHFR. Recently a high resolution crystal structure of R67 DHFR has been solved. From this structure, R67 DHFR is a homotetramer that possesses exact 222 symmetry and a single active site pore that traverses the length of the protein (Narayana et al., 1995). Although this symmetry implies that four symmetry related binding sites must exist for each substrate, isothermal titration calorimetry studies indicate only two molecules bind. Three possible combinations of bound ligands have been observed. These include two dihydrofolate molecules or two NADPH molecules or one substrate + one cofactor (Bradrick et al., 1996). The latter is the productive ternary complex. To date a crystal structure of this ternary complex has been solved. Computational docking studies, however have been used to develop a model of the productive ternary complex (Howell et al., 2001). This model has implicated several active site residues to be involved in ligand binding. Because of the unusual 222 symmetry of this enzyme and the fact it shares no structural similarities with the chromosomal enzyme, R67 DHFR must utilize a different strategy for ligand binding and catalysis. The research in this dissertation has been focused on utilizing site directed mutagenesis as a means to probe the function of active residues implicated by the computational studies to be important in ligand binding and catalysis. Another important goal of this work has been to probe the role interligand cooperativity iv may play in the catalytic function of R67 DHFR. The results of the research presented in this support a model where R67 DHFR utilizes a an unusual “hot spot” binding surface capable of binding both ligands and facilitates catalysis simply by binding ligands in the appropriate orientation to stabilize the transition state. Thus R67 DHFR has adopted a novel yet simple strategy to reach the transition state compared with other more highly evolved DHFRs. v