Structural features of 4-amino antifolates required for substrate activity with mammalian folylpolyglutamate synthetase.
The activity of a series of folic acid analogues 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 decreasing 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: 1) 7-hydroxymethotrexate was a substrate for this enzyme; 2) substrate activity and substrate inhibition were observed with CB 3717, a potent inhibitor of thymidylate synthase; 3) 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 4) leucovorin and methotrexate appeared to be substrates for the same synthetase, but leucovorin 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.
D000588:Amines; D000818:Animals; D005260:Female; D005493:Folic Acid Antagonists / Q000302:isolation & purification / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008815:Mice, Inbred Strains; D010453:Peptide Synthases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D000588:Amines; D005493:Folic Acid Antagonists; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase