We report the enhanced inhibitory potency of
methotrexate (MTX) polyglutamates and
dihydrofolate pentaglutamate on the catalytic activity of phosphoribosylaminoimidazolecarboxamide (
AICAR) transformylase purified from MCF-7 human
breast cancer cells. In the present work, MTX (4-amino-10-methylpteroylglutamic acid) and
dihydrofolate, both monoglutamates, were found to be weak competitive inhibitors of
AICAR transformylase with Kis of 143 and 63 microM, respectively, and their inhibitory capacity was largely unaffected by the glutamated state of the
folate cosubstrate. In contrast, MTX polyglutamates were found to be potent competitive inhibitors, with an approximately 10-fold increase in inhibitory potency with the addition of each
glutamate group up to four (i.e., the pentaglutamate derivative). MTX tetra-and pentaglutamates were the most potent, with equivalent Kis of 5.6 X 10(-8) M or 2500-fold more potent than MTX.
Dihydrofolate pentaglutamate was as potent an inhibitor as MTX pentaglutamate, with a Ki of 4.3 X 10(-8) M. The potent inhibitory effects demonstrated by the
polyglutamate compounds when tested against the
folate monoglutamate substrate were sharply curtailed when
folate pentaglutamate was used as the substrate. MTX and
dihydrofolate pentaglutamates were only 7- and 25-fold more potent than their monoglutamate counterparts under these conditions. A model depicting these complex interactions is postulated. These findings have significant implications regarding the mechanism of action of MTX.