Catalyzes conversion of folates to polyglutamate derivatives allowing concentration of folate compounds in the cell and the intracellular retention of these cofactors, which are important substrates for most of the folate-dependent enzymes that are involved in one-carbon transfer reactions involved in purine, pyrimidine and amino acid synthesis. Dihydrofolate, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 10-formyltetrahydrofolate and 5-formyltetrahydrofolate are the best substrates. Folic acid and 5-methyltetrahydrofolate can also act as substrates. (6S)-5,6,7,8-tetrahydrofolyl-(gamma-L-Glu)(n) + ATP + L-glutamate = (6S)-5,6,7,8-tetrahydrofolyl-(gamma-L-Glu)(n+1) + ADP + H(+) + phosphate A monovalent cation. Inhibited by ammonium sulfate. Inhibited by pentaglutamate derivative of DDATHF, but isoform 2 is inhibited to a greater extent at lower concentrations of the compound that is isoform 5. Isoform 5 is virtually unaffected by H(4)PteGlu(5) and 5,10-CH(2)-H(4)PteGlu(5) at concentrations that substantially inhibits the activity of isoform 2. Isoform 2 and 5 are equally sensitive to polyglutamates of 10-CHO-H(4)-PteGlu. Optimum pH is 8.2-9.5 using 500 uM folic acid (PteGlu) and 25 uM dl-5-formyltetrahydrofolate (dl-5-CHO-H(4)PteGlu) as substrates. Optimum temperature is 37 degrees Celsius. Activity completely lost within a few hours at 4 degrees Celsius in the absence of ATP and glycerol. Cofactor biosynthesis; tetrahydrofolylpolyglutamate biosynthesis. Monomer. With non-specific probe, highest content in kidney and liver and lowest in spleen, lung and small intestine, and readily detectable in all of the tumors except hepatoma. Isoform 1 and isoform 2 expressed in leukemic cells and isoform 4 and isoform 5 in liver cells. Isoform 1 and isoform 2 exclusively expressed in hepatoma and Lewis lung carcinoma. Isoform 1 and isoform 2 also expressed in bone marrow, small intestine and spleen. Kidney expresses isoform 1, isoform 2, isoform 4 and isoform 5. Produced by alternative initiation at Met-43 of isoform 1. Produced by alternative splicing of isoform 1. Produced by alternative promoter usage. Produced by alternative initiation at Met-24 of isoform 4. Belongs to the folylpolyglutamate synthase family. Truncated N-terminus.