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 propertie s of human dihydrofolate reductase enzyme with mutations at position 2 2. Leu-22 was changed to isoleucine, methionine, phenylalanine, and ty rosine to generate the various mutant enzymes. The overall catalytic e fficiency (k(cat)/K-m) for methionine and phenylalanine mutants was re duced similar to 3-fold and > 6-fold for isoleucine and tyrosine mutan ts. An arginine mutant (L(22)R) was also expressed but had a dramatica lly reduced catalytic potential (k(cat) > 250-fold lower than wild-typ e) and therefore was not studied in detail. The K-i for antifolates, m ethotrexate, aminopterin, and trimetrexate are more dramatically affec ted (increased) than the K-m for dihydrofolate, particularly for pheny lalanine and tyrosine mutants. One remarkable feature is that the phen ylalanine mutant is as potently inhibited by piritrexim as is the wild -type human enzyme, although the K-i values for methotrexate and amino pterin were increased 88- and 118-fold, respectively. This is likely r elated to different positioning of the methoxyphenyl side chain of pir itrexim relative to the side chains of other compounds tested. A Chine se hamster cell line harboring the L(22)F mutant also demonstrated an increased sensitivity to piritrexim relative to antifolates.