Variants of human dihydrofolate reductase with substitutions at Leucine-22: Effect on catalytic and inhibitor binding properties

We investigated the enzyme kinetic and antifolate inhibitory properties of human dihydrofolate reductase enzyme with mutations at position 22. Leu-22 was changed to isoleucine, methionine, phenylalanine, and tyrosine to generate the various mutant enzymes. The overall catalytic efficiency (k_cat/K_m) for methionine and phenylalanine mutants was reduced ∼3-fold and >6-fold for isoleucine and tyrosine mutants. An arginine mutant (L²²R) was also expressed but had a dramatically reduced catalytic potential (k_cat >250-fold lower than wild-type) and therefore was not studied in detail. The K_l for antifolates, methotrexate, aminopterin, and trimetrexate are more dramatically affected (increased) than the K_m for dihydrofolate, particularly for phenylalanine and tyrosine mutants. One remarkable feature is that the phenylalanine mutant is as potently inhibited by piritrexim as is the wild-type human enzyme, although the K_l values for methotrexate and aminopterin were increased 88-and 118-fold, respectively. This is likely related to different positioning of the methoxyphenyl side chain of piritrexim relative to the side chains of other compounds tested. A Chinese hamster cell line harboring the L²²F mutant also demonstrated an increased sensitivity to piritrexim relative to antifolates.