Chiericatti G, Santi D V
Department of Biochemistry, University of California, San Francisco 94143-0448, USA.
Biochemistry. 1998 Jun 23;37(25):9038-42. doi: 10.1021/bi9802770.
Structural studies indicate that Asp 221 of Lactobacilluscasei thymidylate synthase forms a hydrogen bond network with the 2-amino and 3-imino groups of the folate [Matthews, D. A. (1990) J. Mol. Biol. 214, 937-948; Finer-Moore, J. S. (1990)Biochemistry 29, 6977-6986] that has been proposed to participate in catalysis. We prepared a complete replacement set of 19 mutants at position 221 of L. casei thymidylate synthase. Of these, the only one with sufficient activity to complement growth of a thymidylate synthase-deficient host was the Cys mutant. To further elucidate the function of the Asp 221 side chain, seven thymidylate synthase 221 mutants were studied in detail with regard to catalysis of dTMP formation and of thymidylate synthase partial reactions. Most of the mutants bound the nucleotide substrate dUMP with only moderate loss of binding affinity, indicating that the Asp side chain does not contribute to dUMP binding. Most of the mutants catalyzed the cofactor-independent dehalogenation of 5-bromodUMP; hence, the Asp side chain of TS is not essential for addition of the catalytic Cys residue to the nucleotide substrate. Mutants showed decreased affinity for the folate cofactor, but those with side chains capable of hydrogen bond formation were less severely affected. Some of the mutants were capable of forming covalent thymidylate synthase-5-fluorodUMP-methylenetetrahydrofolate complex; hence, the Asp side chain is not essential for steps leading to the covalent complex. We conclude that the hydrogen bond network between Asp 221 and the folate cofactor contributes to cofactor binding and a catalytic step after formation of the covalent ternary complex intermediate.
结构研究表明，干酪乳杆菌胸苷酸合酶的天冬氨酸221与叶酸的2-氨基和3-亚氨基形成氢键网络[马修斯，D.A.（1990年）《分子生物学杂志》214卷，937 - 948页；芬纳 - 摩尔，J.S.（1990年）《生物化学》29卷，6977 - 6986页]，该网络被认为参与催化作用。我们制备了干酪乳杆菌胸苷酸合酶221位的19个突变体的完整替换集。其中，唯一具有足够活性以补充胸苷酸合酶缺陷宿主生长的是半胱氨酸突变体。为了进一步阐明天冬氨酸221侧链的功能，对7个胸苷酸合酶221突变体在dTMP形成催化以及胸苷酸合酶部分反应方面进行了详细研究。大多数突变体与核苷酸底物dUMP结合时，结合亲和力仅适度丧失，这表明天冬氨酸侧链对dUMP结合没有贡献。大多数突变体催化5 - 溴dUMP的辅因子非依赖性脱卤反应；因此，胸苷酸合酶的天冬氨酸侧链对于催化性半胱氨酸残基与核苷酸底物的结合不是必需的。突变体对叶酸辅因子的亲和力降低，但那些具有能够形成氢键侧链的突变体受影响较小。一些突变体能够形成共价的胸苷酸合酶 - 5 - 氟dUMP - 亚甲基四氢叶酸复合物；因此，天冬氨酸侧链对于形成共价复合物的步骤不是必需的。我们得出结论，天冬氨酸221与叶酸辅因子之间的氢键网络有助于辅因子结合以及共价三元复合物中间体形成后的催化步骤。
