Purification and properties of Neurospora folylpolyglutamate synthetase

Abstract

Folylpolyglutamate synthetase (FPGS) of Neurospora crassa, wild type (FGSC 853) was purified over 4000-fold and partially stabilized in the presence of glycerol. The purification protocol involved (NH 4) 2SO 4 fractionation and chromatography on DE-52 cellulose, Sephacryl S 200, phenylagarose and hydroxylapatite. Enzyme activity was assayed by incorporation of [ 3H] glutamate into polyglutamates of tetrahydrofolate and 5,10-methylenetetrahydrofolate. The enzyme had properties like those previously reported for the synthetases of bacterial and mammalian origins. Thus, Neurospora FPGS had an apparent M r of ca 65 000, polyglutamate synthesis was favoured at alkaline pH and the reaction required ATP ( K m 286 μM) and magnesium. A variety of folate substrates were utilized with 5,10-methylenetetrahydrofolate monoglutamate being the most effective ( K m 27 μM). The antifolates aminopterin and methotrexate, supplied at 5–100 mM, were not substrates of the enzyme. HPLC analysis of the folate reaction products showed that methylene folate was converted to polyglutamates that were more highly conjugated when the FPGS reaction time was extended to 12 hr. Based on car☐ypeptidase treatment of these products and their behaviour on HPLC it is concluded that purified Neurospora FPGS synthesizes a number of folyloligo-γ-polyglutamates from methylenetetrahydrofolate monoglutamate.