Mutations far from the center of chemical activity in dihydrofolate reductase (DHFR) can affect several steps in the catalytic cycle. Mutations at highly conserved positions and the distal distance of the catalytic center (Met-42, Thr-113, and Gly-121) were designed, including single-point and double-point mutations. Upon ligand binding, the fluorescence of the intrinsic optical probe, tryptophan, decreases due to either fluorescence quenching or energy transfer. We demonstrated an optical approach in measuring the equilibrium dissociation constant for enzyme-cofactor, enzyme-substrate, and enzyme-product complexes in wildtype DHFR and each mutant. We propose that the effects of these distal mutations on ligand... More
Mutations far from the center of chemical activity in dihydrofolate reductase (DHFR) can affect several steps in the catalytic cycle. Mutations at highly conserved positions and the distal distance of the catalytic center (Met-42, Thr-113, and Gly-121) were designed, including single-point and double-point mutations. Upon ligand binding, the fluorescence of the intrinsic optical probe, tryptophan, decreases due to either fluorescence quenching or energy transfer. We demonstrated an optical approach in measuring the equilibrium dissociation constant for enzyme-cofactor, enzyme-substrate, and enzyme-product complexes in wildtype DHFR and each mutant. We propose that the effects of these distal mutations on ligand-binding affinity stem from the spatial steric hindrance, the disturbance on the hydrogen network, or the modification of the protein flexibility. The modified N-terminus tag in DHFR acts as a cap on the entrance of the substrate-binding cavity, squeezes the adenosine binding subdomain, and influences the binding of NADPH in some mutants. If the mutation positions are away from the N-terminus tag and the adenosine binding subdomain, the additive effects due to the N-terminus tag were not observed. In the double-mutant-cycle analysis, double mutations show nonadditive properties upon either cofactor or substrate binding. Also, in general, the first point mutation strongly affects the ligand binding compared to the second one.
