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Folate Analogues as Substrates of Mammalian Folylpolyglutamate Synthetase¹

Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada

The antifolate drugs methotrexate (MTX) and aminopterin (AM) have been tested as substrates for folylpolyglutamate synthetase (FPGS) partially purified from beef liver. The K_m for MTX is 100 µM, and that for AM is 25 µM. These values are considerably higher than those for either tetrahydrofolate or folinic acid. Based on their ratios of V_max to K_m, AM is a better substrate than is MTX for the beef liver FPGS. Both are poorer substrates than tetrahydrofolate. The 7-hydroxy metabolites of MTX and AM also are substrates for FPGS. The reactivity of 7-hydroxymethotrexate is similar to that of MTX, but 7-hydroxyaminopterin is a poorer substrate than AM.

Folinic acid, often used as the rescue agent in high-dose MTX therapy, has a low K_m with mammalian FPGS (7 µM). Its activity is comparable to that of the best substrate, tetrahydrofolate. Low concentrations of folinic acid prevent the formation of polyglutamates of MTX. This inhibition is competitive, presumably because folinic acid and MTX are competing substrates for FPGS.

The activities of folate and antifolate substrates also have been determined with rat liver FPGS. With near-saturating concentrations of AM, MTX, or 7-hydroxymethotrexate, the reaction velocity exceeds that with an optimal concentration of tetrahydrofolate. However, the K_m values of the folate analogues all are greater than those of the tetrahydrofolate coenzymes.

In contrast to the formation of long-chain polyglutamates observed when tetrahydrofolate or folinic acid was the substrate, beef liver FPGS, under our reaction conditions, cannot catalyze the formation from MTX monoglutamate of polyglutamates longer than the triglutamate. MTX di- and triglutamates are poorer substrates than is MTX itself. Longer polyglutamates of MTX, while having no activity as substrates, must bind to the enzyme, because they are inhibitors.

Our observations using MTX and AM with the enzymatic FPGS system help to rationalize the therapeutic use of antifolates.

¹ This investigation was supported by a research grant from the Medical Research Council of Canada and by the Ruhr-Universitat Bochum.

² Student from the Faculty of Medicine, University of Bochum, Bochum, West Germany.

³ Recipient of support from a University of Toronto open fellowship.

⁴ To whom requests for reprints should be addressed.

Received 9/24/84. Revised 3/27/85. Accepted 4/ 3/85.

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