MECHANISTIC STUDIES OF R67 DIHYDROFOLATE-REDUCTASE - EFFECTS OF PH AND AN H62C MUTATION

R67 dihydrofolate reductase (DHFR) is encoded by an R-plasmid, and exp ression of this enzyme in bacteria confers resistance to the antibacte rial drug, trimethoprim, This DHFR variant is not homologous in either sequence or structure with chromosomal DHFRs. The crystal structure o f tetrameric R67 DHFR indicates a single active site pore that travers es the length of the molecule (Narayana, N., Matthews, D. A., Howell, E. E., and Xuong, N.-H. (1995) Not. Struct. Biol. 2, 1018-1025), A PH profile of enzyme activity in R67 DHFR displays an acidic pK(a) that i s protein concentration-dependent. This pK(a) describes dissociation o f active tetramer into two relatively inactive dimers upon protonation of His-62 and the symmetry-related His-162, His-262, and His-362 resi dues at the dimer-dimer interfaces, Construction of an H62C mutation r esults in stabilization of the active tetramer via disulfide bond form ation at the dimer-dimer interfaces. The oxidized, tetrameric form of H62C R67 DHFR is quite active at pH 7, and a pH profile displays incre asing activity at low pH. These results indicate protonated dihydrofol ate (pK(a) = 2.59) is the productive substrate and that R67 DHFR does not possess a proton donor.