Synthesis of (6R)- and (6S)-5,10-dideazatetrahydrofolate oligo-gamma-glutamates: Kinetics of multiple glutamate ligations catalyzed by folylpoly-gamma-glutamate synthetase

摘要

Folylpoly-gamma-glutamate synthetase (FPGS,EC 6.3.2.17) catalyzes the ATP-dependent ligation of glutamic acid to reduced folates including (6S)-5,6,7,8-tetrahydrofolate (H_4PteGlu),as well as to anticancer drugs such as 5,10-dideaza-5,6,7,8-tetrahydrofolate ((6R)-DDAH_4PteGlu_1,(6R)-DDATHF,Lometrexol~(TM)).Synthesis of unlabeled mono- and polyglutamates,DDAH_4PteGlu_n (6R,n=1-6;65,n=1-2),as well as (6R)-DDAH_4Pte[~(14)C]Glu_1,was effected from (6R)- or (65)-5,10-dideazatetrahydropteroyl azide and glutamic acid,H-Glu-gamma-Glu_1,-gamma-Glu-OH (n=0-4),or [~(14)C]glutamic acid,respectively.These compounds were evaluated as FPGS substrates to determine steady-state kinetic constants.Michaelis-Menten kinetics were observed for (6R)-DDAH_4PteGlu_1,the isomer corresponding to H_4PteGlu,whereas marked substrate inhibition was observed for (6S)-DDAH_4PteGlu_n (n=1-2) and (6R)-DDAH_4PteGlu_n (n=2-5),but not (6R)-DDAH_4PteGlu_6.Multiple ligation of glutamate renders a quantitative analysis of these data difficult.However,approximate values of K_M=0.65-1.6 muM and K_1=144-417 muM for DDAH_4PteGlu_n were obtained using a simple kinetic model.