Albrecht A M, Pearce F K, Hutchison D J
J Bacteriol. 1968 May;95(5):1779-89. doi: 10.1128/jb.95.5.1779-1789.1968.
The methylenetetrahydrofolate dehydrogenase of the amethopterin-resistant strain Streptococcus faecium var. durans A(k) was purified 100-fold. Because it is extremely labile, this enzyme required protection by 1 mm nicotinamide adenine dinucleotide phosphate (NADP(+)) during purification; 0.01 mm EADP(+) with 0.1% bovine plasma albumin stabilized the purified enzyme during storage at -20 C. Although the enzyme has properties of sulfhydryl enzymes, thiol compounds were not stabilizers. Oxidation of methylenetetrahydrofolate, catalyzed by the purified enzyme preparation, is NADP(+)-specific and yields methenyltetrahydrofolate and the reduced pyridine nucleotide. K(m) values for NADP(+) and for 5,10-methylenetetrahydrofolate (prepared as the formaldehyde adduct of biologically synthesized l,l-tetrahydrofolate) were calculated to be 0.021 and 0.026 mm, respectively. Neither purine bases and their derivatives nor serine inhibited the reaction. In growing cultures, the differential rate of synthesis of the methylenetetrahydrofolate dehydrogenase was dependent upon the composition of the medium. A medium which contained acid-hydrolyzed casein, and thus an exogenous source of serine, was repressive for this enzyme. In a serine-free, completely defined medium, the amount of folate added (for serine synthesis de novo) affected the duration of the initial exponential growth phase. At the termination of this phase, which primarily reflected the onset of a decreased rate of serine biosynthesis, synthesis of the methylenetetrahydrofolate dehydrogenase was derepressed. Exogenous serine in the completely defined medium prevented the derepression. Furthermore, physiological concentrations of l-serine were repressive not only for the dehydrogenase but also for the methenyltetrahydrofolate cyclohydrolase and the serine hydroxymethyl-transferase. Concomitantly, the differential rate of synthesis of the formyltetrahydrofolate synthetase of S. faecium var. durans A(k) was increased. Apparently, serine regulates the differential rates of syntheses of these enzymes.
对耐氨甲蝶呤的粪肠球菌耐久亚种A(k)的亚甲基四氢叶酸脱氢酶进行了100倍的纯化。由于该酶极其不稳定，在纯化过程中需要1 mM烟酰胺腺嘌呤二核苷酸磷酸（NADP(+)）的保护；0.01 mM EADP(+)与0.1%牛血浆白蛋白在-20℃储存期间可稳定纯化后的酶。尽管该酶具有巯基酶的性质，但硫醇化合物不是稳定剂。纯化酶制剂催化的亚甲基四氢叶酸氧化是NADP(+)特异性的，产生次甲基四氢叶酸和还原型吡啶核苷酸。计算得出NADP(+)和5,10-亚甲基四氢叶酸（作为生物合成的l,l-四氢叶酸的甲醛加合物制备）的K(m)值分别为0.021和0.026 mM。嘌呤碱及其衍生物和丝氨酸均不抑制该反应。在生长培养物中，亚甲基四氢叶酸脱氢酶的合成差异速率取决于培养基的组成。含有酸水解酪蛋白从而含有丝氨酸外源来源的培养基对该酶具有抑制作用。在无丝氨酸的完全确定培养基中，添加的叶酸量（用于从头合成丝氨酸）影响初始指数生长期的持续时间。在该阶段结束时，这主要反映了丝氨酸生物合成速率下降的开始，亚甲基四氢叶酸脱氢酶的合成去抑制。完全确定培养基中的外源丝氨酸阻止了去抑制。此外，生理浓度的l-丝氨酸不仅对脱氢酶有抑制作用，而且对次甲基四氢叶酸环水解酶和丝氨酸羟甲基转移酶也有抑制作用。同时，粪肠球菌耐久亚种A(k)的甲酰四氢叶酸合成酶的合成差异速率增加。显然，丝氨酸调节这些酶的合成差异速率。
