Multiple conformers in active site of human dihydrofolate reductase F31R/Q35E double mutant suggest structural basis for methotrexate resistance.

ABSTRACT: Methotrexate is a slow, tight-binding, competitive inhibitor of human dihydrofolate reductase (hDHFR), an enzyme that provides key metabolites for nucleotide biosynthesis. In an effort to better characterize ligand binding in drug resistance, we have previously engineered hDHFR variant F31R/Q35E. This variant displays a >650-fold decrease in methotrexate affinity, while maintaining catalytic activity comparable to the native enzyme. To elucidate the molecular basis of decreased methotrexate affinity in the doubly substituted variant, we determined kinetic and inhibitory parameters for the simple variants F31R and Q35E. This demonstrated that the important decrease of methotrexate affinity in variant F31R/Q35E is a result of synergistic effects of the combined substitutions. To better under

SUBMITTER: Volpato JP 

PROVIDER: S-EPMC2740434 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature