Liu L, Santi D V
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8604-8. doi: 10.1073/pnas.90.18.8604.
The conserved Asn-229 (N229) of thymidylate synthase (TS, EC 2.1.1.45) provides the only side chain that directly hydrogen bonds with the pyrimidine ring of the substrate dUMP. The carboxamide moiety forms a cyclic hydrogen bond network with the NH-3 and O-4 of the base and is a prime candidate for assisting proton-transfer reactions that occur at O-4 of the pyrimidine ring of dUMP. A complete replacement set of mutants at position 229 of Lactobacillus casei TS (N229 mutants) has been prepared, purified, and characterized. Fifteen of the 19 TS mutants were catalytically active. Steady-state kinetic parameters of N229 mutants varied 17- and 115-fold in the Km values for 5,10-methylene-5,6,7,8-tetrahydrofolate and dUMP, respectively, 1000-fold in kcat values, and 10,000-fold in kcat/Km values. Wild-type TS possesses lower Km and higher kcat and kcat/Km values than any of the TS N229 mutants. We conclude that N229 contributes to, but is not essential for, binding and catalysis. When the wild-type enzyme was not considered, there were excellent correlations between log kcat and the hydrophobicity of the side chains at position 229, in which the more hydrophobic side chains showed higher values. Our results suggest a unique interaction between N229 and the substrates that seems important in appropriately positioning the uracil heterocycle for catalysis. We propose that in the absence of N229, the electrophilic catalyst that transfers protons to the O-4 and stabilizes enol intermediates is a highly conserved molecule of water.
胸苷酸合成酶（TS，EC 2.1.1.45）中保守的天冬酰胺-229（N229）提供了唯一一条直接与底物dUMP嘧啶环形成氢键的侧链。羧酰胺部分与碱基的NH-3和O-4形成环状氢键网络，是协助在dUMP嘧啶环O-4处发生的质子转移反应的主要候选者。已制备、纯化并表征了干酪乳杆菌TS第229位的完整突变体替代组（N229突变体）。19个TS突变体中有15个具有催化活性。N229突变体的稳态动力学参数在5,10-亚甲基-5,6,7,8-四氢叶酸和dUMP的Km值中分别变化了17倍和115倍，在kcat值中变化了1000倍，在kcat/Km值中变化了10000倍。野生型TS比任何TS N229突变体都具有更低的Km值、更高的kcat和kcat/Km值。我们得出结论，N229对结合和催化有贡献，但不是必需的。当不考虑野生型酶时，log kcat与第229位侧链的疏水性之间存在良好的相关性，其中疏水性越强的侧链显示的值越高。我们的结果表明N229与底物之间存在独特的相互作用，这似乎对将尿嘧啶杂环正确定位以进行催化很重要。我们提出，在没有N229的情况下，将质子转移到O-4并稳定烯醇中间体的亲电催化剂是一个高度保守的水分子。
