Tight binding of folate substrates and inhibitors to recombinant mouse glycinamide ribonucleotide formyltransferase
Tight binding of folate substrates and inhibitors to recombinant mouse glycinamide ribonucleotide formyltransferase
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DOI：
10.1021/bi970825u
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发表时间：
1997-08-26
期刊：
影响因子：
2.9
通讯作者：
Moran, RG
中科院分区：
文献类型：
--
作者：
Sanghani, SP;Moran, RG
嘌呤从头合成的原型叶酸抑制剂 5,10-二脱氮四氢叶酸 (DDATHF) 及其六谷氨酸与重组三功能小鼠甘氨酰胺核糖核苷酸甲酰基转移酶 (rmGARFT) 的结合通过平衡透析和稳态动力学研究，使用允许初始速率计算的灵敏测定法。rmGARFT 在感染重组杆状病毒的昆虫细胞中表达，并通过两步程序，每升培养物可生产约 25 毫克纯蛋白质。 DDATHF 与 GARFT 的结合比之前报道的紧密约 50 倍，K-d 和 K-i 值为 2-9 nM，使得该抗叶酸剂的母体形式成为紧密结合抑制剂。DDATHF 的六谷氨酸与 rmGARFT 的结合具有 0.1-0.3 nM 的 K-d 和 K-i 值，这与多谷氨酸化增强抗叶酸剂与 GARFT 结合的观点一致。加夫特。使用单谷氨酸或六谷氨酸底物的动力学分析并未得出 DDATHF 六谷氨酸形式的 K-i 值不同，这与之前的报道相矛盾。通常用于研究 GARFT 的叶酸底物 10-甲酰基-5,8-二脱氮杂叶酸及其六谷氨酸被发现具有非常低的 K-m 值，分别为 75 和 7.4 nM，以及这些底物的叶酸反应产物是同样有效的抑制剂，结果修改了先前动力学实验的解释。产品类似物 DDATHF 和 β-甘氨酰胺核糖核苷酸在存在和不存在另一种的情况下同样与酶结合，这一观察结果与 GARFT 遵循底物有序顺序结合的概念不同。我们得出的结论是，小鼠 GARFT 的动力学与底物结合的随机顺序最为一致，抑制剂 DDATHF 和叶酸底物都是紧密结合的配体，并且聚谷氨酸形式将底物和抑制剂的亲和力增强一个数量级。
The binding of the prototypical folate inhibitor of de novo purine synthesis, 5,10-dideazatetrahydrofolate (DDATHF), and its hexaglutamate to recombinant trifunctional mouse glycinamide ribonucleotide formyltransferase (rmGARFT) was studied by equilibrium dialysis and by steady-state kinetics using sensitive assays that allowed initial rate calculations, rmGARFT was expressed in insect cells infected with a recombinant baculovirus and purified by a two-step procedure that allowed production of about 25 mg of pure protein/L of culture. The binding of DDATHF to GARFT was approximate to 50-fold tighter than previously reported, with K-d and K-i values of 2-9 nM, making the parent form of this antifolate a tight-binding inhibitor, The binding of the hexaglutamate of DDATHF to rmGARFT had K-d and K-i values of 0.1-0.3 nM, consistent with the view that polyglutamation enhances binding of antifolates to GARFT. Kinetic analyses using either mono-or hexaglutamate substrate did not yield different values for the K-i for the hexaglutamate form of DDATHF, in contradiction with previous reports, Both the folate substrate commonly used to study GARFT, 10-formyl-5,8-dideazafolate, and its hexaglutamate were found to have very low K-m values, namely, 75 and 7.4 nM, respectively, and the folate reaction products for these substrates were equally potent inhibitors, results which modify the interpretation of previous kinetic experiments. The product analog DDATHF and beta-glycinamide ribonucleotide bound to enzyme equally well in the presence and absence of the other, an observation at variance with the concept that GARFT obeys an ordered sequential binding of the substrates. We conclude that the kinetics of mouse GARFT are most consistent with a random order of substrate binding, that both the inhibitor DDATHF and the folate substrate are tight-binding ligands, and that polyglutamate forms enhance the affinity of both substrate and inhibitor by an order of magnitude.