STRUCTURAL FEATURES OF 4-AMINO ANTIFOLATES REQUIRED FOR SUBSTRATE ACTIVITY WITH MAMMALIAN FOLYLPOLYGLUTAMATE SYNTHETASE

  • 1 January 1985
    • journal article
    • research article
    • Vol. 27  (1) , 156-166
Abstract
The activity of a series [potentially antineoplastic] of folic acid analogs as substrates for partially purified mouse liver folylpolyglutamate synthetase was determined and the effects of substituents on the binding to, and catalytic processes of, this enzyme were inferred. A 4-amino group improved substrate activity primarily by dereasing the apparent Km while N10-methyl substitution substantially diminished utilization as a substrate, again, by effects on Km. Isosteric replacement of N-10 altered substrate activity. A free .alpha.-carboxyl group in the amino acid side chain was required for catalysis as was the presence of the side chain amide carbonyl group. Modification of the amino acid side chain length profoundly affected activity. Several observations were made that may be relevant to chemotherapy with folate antimetabolites: 7-hydroxymethotrexate was a substrate for this enzyme; substrate activity and substrate inhibition were observed with CB 3717 [N-[p-[N-(2-amino-4(3H)-oxoquinazolin-6-yl)methyl-N-propargyl]amino]benzoyl-L-glutamic acid], a potent inhibitor of thymidylate synthase; potent classical dihydrofolate reductase inhibitors were identified that were either not substrates for mouse liver folylpolyglutamate synthetase (e.g., 4-amino-4-deoxy-N10-methylpteroyl-L-.alpha.-aminoadipate) or were much better substrates than methotrexate for this enzyme (e.g., aminopterin); and leukovorin and methotrexate appeared to be substrates for the same synthetase, but leukovorin saturated the reaction at much lower concentrations. These results have implications for the design of folylpolyglutamate synthetase inhibitors and for the selection of dihydrofolate reductase inhibitors that are either not polyglutamated or are efficiently polyglutamated in vivo.