Mechanistic studies of R67 dihydrofolate reductase. Effects of pH and an H62C mutation.

R67 dihydrofolate reductase (DHFR) is encoded by an R-plasmid, and expression of this enzyme in bacteria confers resistance to the antibacterial drug, trimethoprim. This DHFR variant is not homologous in either sequence or structure with chromosomal DHFRs. The crystal structure of tetrameric R67 DHFR indicates a single active site pore that traverses the length of the molecule (Narayana, N., Matthews, D. A., Howell, E. E., and Xuong, N.-H. (1995) Nat. Struct. Biol. 2, 1018-1025). A pH profile of enzyme activity in R67 DHFR displays an acidic pKa that is protein concentration-dependent. This pKa describes dissociation of active tetramer into two relatively inactive dimers upon protonation of His-62 and the symmetry-related His-162, His-262, and His-362 residues at the dimer-dimer interfaces. Construction of an H62C mutation results in stabilization of the active tetramer via disulfide bond formation at the dimer-dimer interfaces. The oxidized, tetrameric form of H62C R67 DHFR is quite active at pH 7, and a pH profile displays increasing activity at low pH. These results indicate protonated dihydrofolate (pKa = 2.59) is the productive substrate and that R67 DHFR does not possess a proton donor.