The pathway for de novo biosynthesis of
purine nucleotides contains two one-
carbon transfer reactions catalyzed by
glycinamide ribotide (GAR) and 5-aminoimidazole-4-carboxamide ribotide (
AICAR)
transformylases in which N10-formyltetrahydrofolate is the one-
carbon donor. We have found that the
antifolates methotrexate (MTX) and
piritrexim (PTX) completely block the de novo
purine pathway in mouse
L1210 leukemia cells growing in culture but with only minor accumulations of GAR and
AICAR to less than 5% of the
polyphosphate derivatives of
N-formylglycinamide ribotide (FGAR) which accumulate when the pathway is blocked completely by
azaserine. This
azaserine-induced accumulation of FGAR
polyphosphates is completely abolished by MTX, indicating that inhibition of the pathway is at or before
GAR transformylase (reaction 3; Lyons, S. D., and Christopherson, R. I. (1991) Biochem. Int. 24, 187-197). Three h after the addition of MTX (0.1 microM), cellular 5-phosphoribosyl-1-pyrophosphate has accumulated 3.4-fold while 6-methyl-mercaptopurine riboside (25 microM) induces a 6.3-fold accumulation. These data suggest that amido phosphoribosyltransferase catalyzing reaction 1 of the pathway is the primary site of inhibition. In support of this conclusion, we have found that dihydrofolate-Glu5, which accumulates in MTX-treated cells, is a noncompetitive inhibitor of amido phosphoribosyltransferase with a dissociation constant of 3.41 +/- 0.08 microM for interaction with the
enzyme-
glutamine complex in vitro. Folate-Glu5, MTX-Glu5, PTX, dihydrotriazine benzenesulfonyl
fluoride, and
AICAR also inhibit amido phosphoribosyltransferase.