Galivan J H, Maley G F, Maley F
Biochemistry. 1976 Jan 27;15(2):356-62. doi: 10.1021/bi00647a018.
The binding of deoxynucleoside 5'-monophosphates and various folate derivatives to Lactobacillus casei thymidylate synthetase was measured by equilibrium dialysis. The substrate, deoxyuridylate (dUMP), and the product, thymidylate (dTMP), were bound to the enzyme at a ratio of unity and appeared to compete for the same site. The binding of each was tighter in 50 mM Tris-HCl (pH 7.1) than in 50 mM potassium phosphate (pH 7.0). Folate derivatives increased the affinity of the enzyme for the substrate to a greater extent than for the product, although they themselves did not appear to be bound in the absence of substrate or substrate analogues. However, in the presence of enzyme, dUMP or 4-N-OH-dCMP, and either 7,8-dihydrofolate or methotrexate, a ternary complex was obtained, with the folate derivatives exhibiting single site binding. The binding of dUMP in the ternary complex was 25-fold greater than that of 7,8-dihydrofolate and 50-fold greater than that of methotrexate. Supporting evidence for the enhanced stability of the ternary complex was provided by heat inactivation studies. As in the case of deoxynucleotide binding to the synthetase, the ternary complex was more stable in Tris HCl than in potassium phosphate buffer. The binding characteristics of the substrate analogue 5-fluoro-2'-deoxyuridylate (FdUMP) could be clearly distinguished from that of dUMP by comparing their binding in phphate and Tris-HCl. While each deoxynucleotide exhibited only single site binding in phosphate, a second site was clearly demonstrated for FdUMP with Tris-HCl. The binding of FdUMP to each site appeared to be equal in the presence of methotrexate or (-)5,10-methylene tetrahydrofolate and was increased about 17-fold in Tris-HCl. Two sites were also obtained for FdUMP in the presence of 7,8-dihydrofolate, but Scatchard analyses revealed a biphasic curve, with the second site possessing a higher dissociation constant than the first. A second low affinity FdUMP binding site was also detected in phosphate buffer when 7,8-dihydrofolate or (-)5,10-methylene tetrahydrofolate was included in the binding assay. In the presence of (+)5,10-methylene tetrahydrofolate, however, 2 mol of FdUMP was bound stoichoimetrically to 1 mol of enzyme regardless of the buffer used. The significance of these results is discussed in relation to the presence of two apparently identical subunits in the native enzyme.
通过平衡透析法测定了脱氧核苷5'-单磷酸和各种叶酸衍生物与干酪乳杆菌胸苷酸合成酶的结合情况。底物脱氧尿苷酸（dUMP）和产物胸苷酸（dTMP）以1:1的比例与该酶结合，且似乎竞争相同的位点。在50 mM Tris-HCl（pH 7.1）中，每种物质的结合都比在50 mM磷酸钾（pH 7.0）中更紧密。叶酸衍生物增加该酶对底物的亲和力的程度大于对产物的亲和力，尽管在没有底物或底物类似物的情况下它们本身似乎未被结合。然而，在有酶、dUMP或4-N-OH-dCMP以及7,8-二氢叶酸或甲氨蝶呤存在的情况下，可得到一种三元复合物，其中叶酸衍生物表现出单一位点结合。在三元复合物中，dUMP的结合比7,8-二氢叶酸大25倍，比甲氨蝶呤大50倍。热失活研究为三元复合物稳定性增强提供了支持证据。与脱氧核苷酸与合成酶的结合情况一样，三元复合物在Tris HCl中比在磷酸钾缓冲液中更稳定。通过比较底物类似物5-氟-2'-脱氧尿苷酸（FdUMP）在磷酸盐和Tris-HCl中的结合情况，可以清楚地将其与dUMP的结合特性区分开来。虽然每种脱氧核苷酸在磷酸盐中仅表现出单一位点结合，但在Tris-HCl中FdUMP明显显示出第二个位点。在甲氨蝶呤或（-）5,10-亚甲基四氢叶酸存在的情况下，FdUMP与每个位点的结合似乎相等，且在Tris-HCl中增加约17倍。在7,8-二氢叶酸存在的情况下，FdUMP也出现两个位点，但Scatchard分析显示为双相曲线，第二个位点的解离常数高于第一个。当在结合测定中加入7,8-二氢叶酸或（-）5,10-亚甲基四氢叶酸时，在磷酸盐缓冲液中也检测到第二个低亲和力的FdUMP结合位点。然而，在（+）5,10-亚甲基四氢叶酸存在的情况下，无论使用何种缓冲液，2摩尔的FdUMP都化学计量地结合到1摩尔的酶上。结合天然酶中存在两个明显相同的亚基来讨论这些结果的意义。
