Investigation of mutants in the active site and the β-bulge region of dihydrofolate reductase

Aspartic acid 27 is conserved in all bacterial dihydrofolate reductases (DHFR) and it serves as a proton donor in the catalytic mechanism (Howell et al., Sci 23:1123). Not surprisingly, the D27S mutant protein has a greatly decreased k_cat at pH 7.0. Second site suppressor mutations have been generated in the D27S DHFR gene by genetic selections and site-directed mutagenesis techniques. These mutations partially suppress the effect caused by removal of the proton donor. To investigate whether these individual suppressing mutations have additive effects, a D27S+E17G+D127G+F137S quadruple mutant gene was constructed. The k_cat and K_m (DHF) of the quadruple mutant DHFR are similar to those of the D27S+F137S double mutant, thus an additive effect is not found in the quadruple mutant DHFR. Additionally, the D27S+T113E+F153S and D27S+T113E+F137S and D27S+T113E+I155N triple mutant DHFR genes were generated to test the relationship between the active site and a β-bulge region. The T113E mutation puts a potential proton donating group in the active site, although at a different position. The triple mutants have different mobilities on a nondenaturing polyacrylamide gel when compared to wild-type and D27S DHFRs, indicating altered conformations. The k_cat and K_m values of the D27S+T113E+F153S triple mutant DHFR are slightly better than those of the D27S+T113E double mutant but they are worse than those of the D27S+F153S double mutant DHFR. The triple mutant DHFR does not show additive effects.