DL-3,3-difluoroglutamate: an enhancer of folylpolyglutamate elongation.
The effect of 3,3-difluoroglutamate (F2Glu) on the reaction catalyzed by rat liver folypolyglutamate synthetase was investigated. F2Glu was a potent, concentration-dependent inhibitor of poly(gamma-glutamylation) using [3H]Glu and either methotrexate (4-NH2-10-CH3PteGlu) or tetrahydrofolate as substrates. It was determined that F2Glu acted as an alternate substrate, but in contrast to the previously characterized alternate substrate 4-fluoroglutamate (McGuire, J.J., and Coward, J.K. (1985) J. Biol. Chem. 260, 6747-6754), it did not terminate polyglutamate chain elongation. Instead, F2Glu promoted chain elongation. Thus, synthesis of products from [3H]methotrexate containing 1 and 2 additional amino acid residues occurred at a substantially higher rate in the presence of F2Glu when compared to identical reactions in the presence of Glu; this was more pronounced for the product containing 2 additional residues. Identities of the products were established by their respective chromatographic elution positions and by limit digestion with gamma-glutamyl hydrolases. Ligation of Glu to 4-NH2-10-CH3PteGlu-gamma-(3,3-difluoroglutamate) was also enhanced. These results are consistent with F2Glu enhancing the synthesis of poly(gamma-glutamate) metabolites at the level of either the incoming amino acid (glutamate analog) or the gamma-glutamyl acceptor species. F2Glu is thus the first glutamate analog which enhances chain elongation catalyzed by folypolyglutamate synthetase.
McGuire
J J
JJ
Grace Cancer Drug Center, Roswell Park Memorial Institute, Buffalo, New York 14263.
Haile
W H
WH
Bey
P
P
Coward
J K
JK
D000818:Animals; D005492:Folic Acid / Q000031:analogs & derivatives*; D005971:Glutamates / Q000378:metabolism / Q000494:pharmacology*; D018698:Glutamic Acid; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008727:Methotrexate / Q000378:metabolism; D010453:Peptide Synthases / Q000378:metabolism*; D011624:Pteroylpolyglutamic Acids / Q000096:biosynthesis*; D051381:Rats; D013379:Substrate Specificity
D005971:Glutamates; D011624:Pteroylpolyglutamic Acids; C065405:3,3-difluoroglutamate; D018698:Glutamic Acid; D005492:Folic Acid; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D008727:Methotrexate