| esidues Leu28, Phe31, Ile50 and Leu54 in E. coli DHFR have been studied by site-specific and directed random mutagenesis, and kinetics. Studies suggest that the mutant structures do not change compared to the wild-type.;Two sets of mutants have been constructed. The first set includes two single mutants (L28Y and L54F) and a double mutant (L28Y-L54F). The second set includes two double mutants (L28A-F31A and I50A-L54G) and a quadruple mutant (L28A-F31A-I50A-L54G). L28Y and L54F, and L28A-F31A and I50A-L54G are spacially separated (;Construction of a related mutants set including L28F, L54F, and L28F-L54F provides a means for assessing the interactions of Phe28 and Tyr28 with the substrate and the residue 54 sidechain. It is found that a single functional group change, from Phe C4-H to Tyr C4-OH, can influence the additivity effects. The comparison of nonadditivity for three interrelated double mutational cycles (WT ;Directed random mutagenesis has been applied to the same residues to construct a plasmid library containing 81 new quadruple mutants. A screening method is developed. Mutant L28F-F31V-I50V-L54F is obtained after the screening and characterized. It binds MTX... |
