Enzymatic synthesis of folylpolyglutamates. Characterization of the reaction and its products.

Rat liver folylpolyglutamate synthetase was partially purified and its reaction and products were characterized. The preparation contained no conjugase activity. Gel filtration analysis revealed a molecular weight of 69,000. The synthetase was optimally active at pH 8.4 (37 degrees C), required mercaptoethanol and a monovalent cation, and was highly specific for L-glutamate. Only purine nucleoside triphosphates served as the energy source for the reaction; ATP and dATP gave the best activity. All naturally occurring folates (including 5-methyl-tetrahydrofolic acid) as well as a number of folate analogs (including methotrexate) served as substrates. The unnatural diastereoisomer of at least one folate, 5,6,7,8-tetrahydrofolic acid, was also a substrate. Modifications of the terminal, acceptor glutamate led to loss of substrate activity, as well as loss of binding. High pressure liquid chromatography analysis, conjugase digestion, double radiolabel studies, and amino acid analysis of acid-hydrolyzed product confirmed that folylpoly-gamma-glutamates were synthesized. High concentrations of (dl)-5,6,7,8-tetrahydrofolic acid favored accumulation of short chain (predominantly diglutamate) products while low concentrations favored accumulation of longer chains (predominantly tetraglutamate). This inverse relationship between concentration and chain length may be of regulatory significance. Synthesis of pentaglutamate forms, the predominant chain length of rat liver folates in vivo, was detected at low (dl)-5,6,7,8-tetrahydrofolic acid, but hexaglutamate was not detected. Synthetic (l)-5,6,7,8-tetrahydropteroylpentaglutamate was a poor substrate for the synthetase but it inhibited formation of polyglutamates from monoglutamates. These observations indicate that the predominant chain length of folates in rat liver may be determined solely by the substrate specificity of the rat liver synthetase. Inhibition by the specificity of the rat liver synthetase. Inhibition by the pentaglutamate derivative offers a means by which folylpolyglutamates could regulate their own synthesis.