Studies on the polyglutamate specificity of thymidylate synthase from fetal pig liver.
Studies on the polyglutamate specificity of thymidylate synthase from fetal pig liver.
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DOI：
10.1021/bi00321a091
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发表时间：
1984-12
期刊：
影响因子：
2.9
通讯作者：
Yong Zhi Lu;Patrick D. Aiello;R. Matthews
中科院分区：
文献类型：
--
作者：
Yong Zhi Lu;Patrick D. Aiello;R. Matthews
通过使用 Affigel-Blue、DEAE-52 和羟基磷灰石色谱法，胸苷酸合酶已从胎猪肝脏中纯化了 1700 倍。稳态动力学测量表明催化通过有序的顺序机制进行。当使用 5,10-亚甲基四氢蝶酰单谷氨酸 (CH2-H4PteGlu1) 作为底物时，dUMP 先于 CH2-H4PTeGlu1 结合，7,8-二氢蝶酰单谷氨酸 (H2PteGlu1) 先于 dTMP 释放。蝶酰聚谷氨酸 (PteGlun) 是胸苷酸合酶活性的抑制剂，与 CH2-H4PteGlu1 具有竞争性，但与 dUMP 没有竞争性。抑制常数（Ki 值）对应于 PteGlun 从酶-dUMP-PteGlun 三元复合物中解离的解离常数，已确定具有 1 至 7 个谷氨酰残基的 PteGlun 衍生物：PteGlu1，10 microM； PteGlu2，0.3 微摩尔； PteGlu3，0.2 微摩尔； PteGlu4，0.06 微摩尔； PteGlu5，0.10 微摩尔； PteGlu6，0.12 微摩尔； PteGlu7，0.15 微摩尔。因此，来自胎猪肝脏的胸苷酸合酶优先结合具有四个谷氨酰残基的蝶酰聚谷氨酸，但具有二至七个谷氨酰残基的衍生物的结合强度比单谷氨酸至少强30倍。当 CH2-H4PteGlu4 用作胸苷酸生物合成的一种碳供体时，底物结合和产物释放的顺序相反，CH2-H4PteGlu4 的结合先于 dUMP 的结合，而 dTMP 的释放先于 H2PteGlu4 的释放。随着底物聚谷氨酸链长度的变化，dUMP 和 CH2-H4PteGlun 的 Vmax 和 Km 值变化相对较小。（摘要截断为 250 字）
Thymidylate synthase has been purified 1700-fold from fetal pig livers by using chromatography on Affigel-Blue, DEAE-52, and hydroxylapatite. Steady-state kinetic measurements indicate that catalysis proceeds via an ordered sequential mechanism. When 5,10-methylenetetrahydro-pteroylmonoglutamate (CH2-H4PteGlu1) is used as the substrate, dUMP is bound prior to CH2-H4PTeGlu1, and 7,8-dihydropteroylmonoglutamate (H2PteGlu1) is released prior to dTMP. Pteroylpolyglutamates (PteGlun) are inhibitors of thymidylate synthase activity and are competitive with respect to CH2-H4PteGlu1 and uncompetitive with respect to dUMP. Inhibition constants (Ki values), which correspond to dissociation constants for the dissociation of PteGlun from the enzyme-dUMP-PteGlun ternary complex, have been determined for PteGlun derivatives with one to seven glutamyl residues: PteGlu1, 10 microM; PteGlu2, 0.3 microM; PteGlu3, 0.2 microM; PteGlu4, 0.06 microM; PteGlu5, 0.10 microM; PteGlu6, 0.12 microM; PteGlu7, 0.15 microM. Thus, thymidylate synthase from fetal pig liver preferentially binds pteroylpolyglutamates with four glutamyl residues, but derivatives with two to seven glutamyl residues all bind at least 30-fold more tightly than the monoglutamate. When CH2-H4PteGlu4 is used as the one carbon donor for thymidylate biosynthesis, the order of substrate binding and product release is reversed, with binding of CH2-H4PteGlu4 preceding that of dUMP and release of dTMP preceding release of H2PteGlu4. Vmax and Km values for dUMP and CH2-H4PteGlun show relatively little change as the polyglutamate chain length of the substrate is varied.(ABSTRACT TRUNCATED AT 250 WORDS)