Role of S65, Q67, I68, and I69 residues in homotetrameric R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR) shares no sequence or structural homolog y with chromosomal DHFRs. This enzyme arose recently in response to the cli nical use of the antibacterial drug trimethoprim. R67 DHFR is a homotetrame r possessing a single active site pore. A high-resolution crystal structure shows the homotetramer possesses exact 222 symmetry [Narayana, N., et al. (1995) Nat. Struct. Biol. 2, 1018-1025]. This symmetry dictates four symmet ry-related binding sites must exist for each substrate as well as each cofa ctor. Isothermal titration calorimetry studies, however, indicate only two molecules bind: either two dihydrofolate molecules, two NADPH molecules, or one substrate and one cofactor [Bradrick, T. D., et al. (1996) Biochemistr y 35, 11414-11424]. The latter is the productive tertiary complex. To evalu ate the role of S65, Q67, I68, and Y69 residues, located near the center of the active site pore, site-directed mutagenesis was performed. One mutatio n in the gene creates four mutations per active site pore which typically r esult in large cumulative effects. Steady state kinetic data indicate the m utants have altered K-m values for both cofactor and substrate. For example , the Y69F R67 DHFR displays an 8-fold increase in the K-m for dihydrofolat e and a 20-fold increase in the K-m for NADPH. Residues involved in ligand binding in R67 DHFR display very little, if any, specificity, consistent wi th their possessing dual roles in binding. These results support a model wh ere R67 DHFR utilizes an unusual "hot spot" binding surface capable of bind ing both ligands and indicate this enzyme has adopted a novel yet simple ap proach to catalysis.