Mechanism and function of mammalian folylpoly-gamma-glutamate synthetase

Mammalian Folylpoly-γ-glutamate synthetase (FPGS) adds multiple moles of glutamic acid to the γ-carboxyl group of folates and antifolates. Polyglutamation of folates serves to trap these compounds within the cell for use in nucleotide and amino acid biosynthesis. Thus, intracellular folates exist as tetrahydrofolylpoly-γ-glutamates with the penta and hexa-glutamate forms predominating.;Two isoforms of FPGS that differ by 18 amino acids at their amino terminus are expressed in mouse tissues. One isozyme is expressed in differentiated tissue and the other in all rapidly proliferating cell types. Recombinant mouse cytosolic FPGS isoforms were equally effective in ligating a mole of glutamic acid to folylmonoglutamate substrates. Each isoform was also capable of forming long chain polyglutamate derivatives of 6R-5,10-dideazatetrahydrofolate (6R-DDATHF). The FPGS isoform derived from rapidly proliferating tissue was much more sensitive to inhibition by (6R)-5,10-CH2-H4PteGlu 5 and (6S)-H4PteGlu5 than the isoform expressed in differentiated tissues. The decreased sensitivity of the FPGS expressed in mouse liver and kidney to feedback inhibition by folylpolyglutamates may have evolved to permit accumulation of a larger folate cofactor pool in these tissues than that found in proliferating tissue.;Addition of multiple moles of glutamic acid to folate antimetabolites by human FPGS is required for antifolates to be effective as chemotherapeutic agents. Many antifolates, such as 6R-DDATHF, are rapidly converted to penta and hexaglutamate molecules by recombinant human cytosolic FPGS whereas other antifolates, typified by aminopterin, predominantly form the diglutamate derivative. A time course monitoring the formation of polyglutamates of 6R-DDATHF by FPGS, with stoichiometric amounts of enzyme and substrate, indicated that the formation of the di and triglutamate product was linked and were thus consistent with a processive polyglutamation mechanism. However, when FPGS was presented with increasing amounts of 6R-DDATHF substrate the predominant product was the diglutamate of DDATHF and no formation of triglutamate product was observed; the results were incompatible with a processive mechanism. Steady state kinetics performed with the diglutamate substrates indicated that the kcat of aminopterin diglutamate was 400-fold lower than aminopterin. Additionally, 6R-DDATHF diglutamate, while exhibiting a slightly lower kcat than 6R-DDATHF (1.7-fold), was found to bind to FPGS at a 30 times lower substrate concentration in comparison to 6R-DDATHF. Thus, the results indicated that the kinetics of the diglutamate species of aminopterin and DDATHF dictated the extent to which these compounds were metabolized to higher polyglutamate forms by rhcFPGS.