Schoo M M, Pristupa Z B, Vickers P J, Scrimgeour K G
Cancer Res. 1985 Jul;45(7):3034-41.
The antifolate drugs methotrexate (MTX) and aminopterin (AM) have been tested as substrates for folylpolyglutamate synthetase (FPGS) partially purified from beef liver. The Km for MTX is 100 microM, and that for AM is 25 microM. These values are considerably higher than those for either tetrahydrofolate or folinic acid. Based on their ratios of Vmax to Km, AM is a better substrate than is MTX for the beef liver FPGS. Both are poorer substrates than tetrahydrofolate. The 7-hydroxy metabolites of MTX and AM also are substrates for FPGS. The reactivity of 7-hydroxymethotrexate is similar to that of MTX, but 7-hydroxyaminopterin is a poorer substrate than AM. Folinic acid, often used as the rescue agent in high-dose MTX therapy, has a low Km with mammalian FPGS (7 microM). Its activity is comparable to that of the best substrate, tetrahydrofolate. Low concentrations of folinic acid prevent the formation of polyglutamates of MTX. This inhibition is competitive, presumably because folinic acid and MTX are competing substrates for FPGS. The activities of folate and antifolate substrates also have been determined with rat liver FPGS. With near-saturating concentrations of AM, MTX, or 7-hydroxymethotrexate, the reaction velocity exceeds that with an optimal concentration of tetrahydrofolate. However, the Km values of the folate analogues all are greater than those of the tetrahydrofolate coenzymes. In contrast to the formation of long-chain polyglutamates observed when tetrahydrofolate or folinic acid was the substrate, beef liver FPGS, under our reaction conditions, cannot catalyze the formation from MTX monoglutamate of polyglutamates longer than the triglutamate. MTX di- and triglutamates are poorer substrates than is MTX itself. Longer polyglutamates of MTX, while having no activity as substrates, must bind to the enzyme, because they are inhibitors. Our observations using MTX and AM with the enzymatic FPGS system help to rationalize the therapeutic use of antifolates.
抗叶酸药物甲氨蝶呤（MTX）和氨蝶呤（AM）已被作为从牛肝中部分纯化的叶酰聚谷氨酸合成酶（FPGS）的底物进行测试。MTX的米氏常数（Km）为100微摩尔，AM的Km为25微摩尔。这些值远高于四氢叶酸或亚叶酸的相应值。根据它们的最大反应速度（Vmax）与Km的比值，对于牛肝FPGS而言，AM是比MTX更好的底物。两者都是比四氢叶酸更差的底物。MTX和AM的7 - 羟基代谢产物也是FPGS的底物。7 - 羟基甲氨蝶呤的反应活性与MTX相似，但7 - 羟基氨蝶呤是比AM更差的底物。亚叶酸常用于大剂量MTX治疗中的解救药物，它与哺乳动物FPGS的Km较低（7微摩尔）。其活性与最佳底物四氢叶酸相当。低浓度的亚叶酸可阻止MTX形成聚谷氨酸。这种抑制是竞争性的，推测是因为亚叶酸和MTX是FPGS的竞争性底物。也已用大鼠肝FPGS测定了叶酸和抗叶酸底物的活性。在AM、MTX或7 - 羟基甲氨蝶呤浓度接近饱和时，反应速度超过了在四氢叶酸最佳浓度时的反应速度。然而，叶酸类似物的Km值均大于四氢叶酸辅酶的Km值。与以四氢叶酸或亚叶酸为底物时观察到的形成长链聚谷氨酸不同，在我们的反应条件下，牛肝FPGS无法催化MTX单谷氨酸形成比三谷氨酸更长的聚谷氨酸。MTX二聚谷氨酸和三聚谷氨酸是比MTX本身更差的底物。MTX更长的聚谷氨酸虽然没有底物活性，但必定会与酶结合，因为它们是抑制剂。我们使用MTX和AM对酶促FPGS系统的观察有助于解释抗叶酸药物的治疗用途。
