Strong W B, Tendler S J, Seither R L, Goldman I D, Schirch V
Department of Biochemistry, Virginia Commonwealth University Medical College of Virginia, Richmond 23298.
J Biol Chem. 1990 Jul 25;265(21):12149-55.
Serine hydroxymethyltransferase and the trifunctional enzyme C1-tetrahydrofolate synthase have been purified to near homogeneity from L1210 cells. Kinetic constants (Km and kcat) have been determined for both folate and non-folate substrates. The effect of increasing glutamate chain length on affinity and catalytic efficiency were determined for the four activities. The studies show that the structural and catalytic properties of the two L1210 enzymes are very similar to the corresponding enzymes purified from rabbit liver. Antibodies to both rabbit serine hydroxymethyltransferase and C1-tetrahydrofolate synthase cross-react with the corresponding L1210 enzymes. The intracellular concentration of active sites of serine hydroxymethyltransferase and C1-tetrahydrofolate synthase in L1210 cells are both 9 microM. The combined concentration of these two enzymes exceeds the previously reported concentration of 10 microM for total intracellular folates. A network thermodynamic computer model of one carbon metabolism (Seither, R. L., Trent, D. F., Mikulecky, D. C., Rape, T. J., and Goldman, I. D. (1989) J. Biol. Chem. 264, 17016-17023) suggests that complete inhibition of cytosolic serine hydroxymethyltransferase would neither significantly decrease the rates of biosynthesis of purines and thymidylate nor significantly alter the rate of interconversion of tetrahydrofolate cofactors to dihydrofolate with subsequent inhibition of dihydrofolate reductase.
丝氨酸羟甲基转移酶和三功能酶C1-四氢叶酸合酶已从L1210细胞中纯化至接近均一状态。已测定了叶酸和非叶酸底物的动力学常数（Km和kcat）。针对这四种活性，测定了增加谷氨酸链长度对亲和力和催化效率的影响。研究表明，这两种L1210酶的结构和催化特性与从兔肝中纯化的相应酶非常相似。兔丝氨酸羟甲基转移酶和C1-四氢叶酸合酶的抗体与相应的L1210酶发生交叉反应。L1210细胞中丝氨酸羟甲基转移酶和C1-四氢叶酸合酶活性位点的细胞内浓度均为9 microM。这两种酶的总浓度超过了先前报道的细胞内总叶酸浓度10 microM。一个一碳代谢的网络热力学计算机模型（Seither, R. L., Trent, D. F., Mikulecky, D. C., Rape, T. J., and Goldman, I. D. (1989) J. Biol. Chem. 264, 17016 - 17023）表明，完全抑制胞质丝氨酸羟甲基转移酶既不会显著降低嘌呤和胸苷酸的生物合成速率，也不会显著改变四氢叶酸辅因子向二氢叶酸的相互转化速率，随后也不会抑制二氢叶酸还原酶。
