Synthesis of (6R)- and (6S)-5,10-dideazatetrahydrofolate oligo-gamma-glutamates: kinetics of multiple glutamate ligations catalyzed by folylpoly-gamma-glutamate synthetase.
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 (H4PteGlu), as well as to anticancer drugs such as 5,10-dideaza-5,6,7,8-tetrahydrofolate ((6R)-DDAH4PteGlu1, (6R)-DDATHF, Lometrexol). Synthesis of unlabeled mono- and polyglutamates, DDAH4PteGlu(n) (6R, n = 1-6; 6S, n = 1-2), as well as (6R)-DDAH4Pte[14C]Glu1, was effected from (6R)- or (6S)-5,10-dideazatetrahydropteroyl azide and glutamic acid, H-Glu-gamma-Glu(n)-gamma-Glu-OH (n = 0-4), or [14C]glutamic acid, respectively. These compounds were evaluated as FPGS substrates to determine steady-state kinetic constants. Michaelis-Menten kinetics were observed for (6R)-DDAH4PteGlu1, the isomer corresponding to H(4)PteGlu, whereas marked substrate inhibition was observed for (6S)-DDAH4PteGlu(n) (n = 1-2) and (6R)-DDAH4PteGlu(n) (n = 2-5), but not (6R)-DDAH4PteGlu6. Multiple ligation of glutamate renders a quantitative analysis of these data difficult. However, approximate values of K(M) = 0.65-1.6 microM and K(I) = 144-417 microM for DDAH4PteGlu(n) were obtained using a simple kinetic model.
Tomsho
John W
JW
Department of Medicinal Chemistry, University of Michigan, 3813 Chemistry, 930 N. University, Ann Arbor, MI 48109-1055, USA.
McGuire
John J
JJ
Coward
James K
JK
D002384:Catalysis; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism*; D011624:Pteroylpolyglutamic Acids / Q000138:chemical synthesis*; D013379:Substrate Specificity
D011624:Pteroylpolyglutamic Acids; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase