McGuire J J, Haile W H, Bey P, Coward J K
Grace Cancer Drug Center, Roswell Park Memorial Institute, Buffalo, New York 14263.
J Biol Chem. 1990 Aug 25;265(24):14073-9.
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.
研究了3,3 - 二氟谷氨酸（F2Glu）对大鼠肝脏叶酰聚谷氨酸合成酶催化反应的影响。以[3H]谷氨酸为原料，甲氨蝶呤（4 - NH2 - 10 - CH3PteGlu）或四氢叶酸为底物时，F2Glu是聚（γ - 谷氨酰化）反应的强效、浓度依赖性抑制剂。已确定F2Glu作为替代底物，但与先前表征的替代底物4 - 氟谷氨酸不同（McGuire, J.J., and Coward, J.K. (1985) J. Biol. Chem. 260, 6747 - 6754），它不会终止聚谷氨酸链的延伸。相反，F2Glu促进链的延伸。因此，与在谷氨酸存在下的相同反应相比，在F2Glu存在下，含有1个和2个额外氨基酸残基的[3H]甲氨蝶呤产物的合成速率显著更高；对于含有2个额外残基的产物，这种情况更为明显。通过各自的色谱洗脱位置和用γ - 谷氨酰水解酶进行极限消化来确定产物的身份。谷氨酸与4 - NH2 - 10 - CH3PteGlu - γ - （3,3 - 二氟谷氨酸）的连接也得到增强。这些结果与F2Glu在进入的氨基酸（谷氨酸类似物）或γ - 谷氨酰受体物种水平上增强聚（γ - 谷氨酸）代谢物的合成一致。因此，F2Glu是第一种增强叶酰聚谷氨酸合成酶催化的链延伸的谷氨酸类似物。
