| Dihydrofolate reductases (DHFR) catalyze the NADPH dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). R67 DHFR is an R-plasmid encoded enzyme that confers resistance to the antibiotic trimethoprim (TMP). Due to the ability of R67 DHFR to avoid enzymatic inhibition in the presence of compounds that severely inhibit other DHFRs, its catalytic mechanism is being investigated.;R67 DHFR is a homotetramer that contains a single active site pore. The only charged residue in each monomer of the active site is K32. The only other charged residue near the active site is K33. NMR and docking studies predict that K32 interacts with the 2' phosphate of NADPH. In addition, docking studies predict that K32 interacts with the tail of DHF. K32M substitutions were generated to examine its role in ionic interactions but result in the formation of an inactive dimer. To test predictions that ionic interactions are involved in ligand binding, salt effects were evaluated on various parameters including Kd(NADPH), qTotal for folate, kcat, kcat/Km(NADPH), and k cat/Km(DHF). Salt sensitivities of these parameters suggest that ionic interactions are involved in ligand binding and catalysis. In addition, steady-state kinetics were performed in the presence of the alternate cofactor NADH. A 50-fold reduction in kcat/Km was observed in the presence of alternate cofactor indicating the 2' phosphate is involved in an ionic interaction with R67 DHFR.;To directly evaluate the role of K32 in ionic interactions in R67 DHFR, a four-gene copy construct of R67 DHFR, which possesses K32M asymmetric mutations was utilized. Mutants that possess a single substitution in each half pore, result in a decrease in kcat. These results suggest that R67 DHFR uses its symmetry to achieve catalysis. The mutant that possesses two substitutions in one-half pore results in a significant increase in KmS and k cat. The increase in kcat observed with this mutant indicates that loss of an ionic interaction facilitates formation of the transition state.;These data support that K32 participates in an ionic contact(s) with NADPH and DHF in the ground state. Loss of one of these interactions facilitates formation of the transition state. |
