Effects of Distal Mutations on Ligand-Binding Affinity in E. coli Dihydrofolate Reductase.

ABSTRACT: 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 ecDHFR and each mutant. We propose that the effects of these distal mutations on ligand-binding affinity stem from the spatial steric hindrance, the disturban

SUBMITTER: Huang CH 

PROVIDER: S-EPMC8515367 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature