CRITICAL ROLE OF PHENYLALANINE-34 OF HUMAN DIHYDROFOLATE-REDUCTASE INSUBSTRATE AND INHIBITOR BINDING AND IN CATALYSIS

Directed mutagenesis has been used to construct five variants of human dihydrofolate reductase in which smaller residues are substituted for phenylalanine 34, a residue participating in the binding of substrate and methotrexate by interaction with their pteridine rings. The varia nt enzymes are stable and have decreased affinities for methotrexate ( by factors of 2700-60000 at pH 7.65) due to a decreased rate of methot rexate association and a much larger increase in the rate constant for dissociation. However, the catalytic efficiencies of the variants are also lowered by factors of 160-5000, so that it is doubtful whether t hese enzymes are capable of conferring methotrexate resistance on the cells harboring them. High concentrations of dihydrofolate cause marke d inhibition of all the variants, which complicates the determination of kinetic parameters. By the use of stopped-flow spectrophotometry an d fluorimetry and other methods, it has been shown that, like the wild -type enzyme, the variants have a branched reaction pathway, but in co ntrast to the wild-type enzyme, the distribution of flux between alter nate pathways is dependent on the concentration of dihydrofolate. This different branch point is a consequence of the very rapid dissociatio n of tetrahydrofolate from the ternary product complexes of the varian t enzymes. Inhibition by dihydrofolate is due to its combination with the enzyme-NADP complex and the slow dissociation of NADP from the