Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase
Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase
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DOI：
10.1093/protein/13.8.557
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发表时间：
2000-08-01
期刊：
影响因子：
--
通讯作者：
Santi, DV
中科院分区：
文献类型：
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作者：
Kawase, S;Cho, SW;Santi, DV
胸苷合成酶(TS，EC 2.1.1.45)中的精氨酸R23、R178、R179和R218是底物Dump的磷酸部分的氢键供体。为了研究这些精氨酸对酶功能的贡献，我们在干酪乳杆菌TS的四个位点上制备了完整的突变体替换集，R23的突变使Dump的K-m增加了2-20倍，辅因子的K-m增加了8-40倍，K(CAT)减少了9-20倍，反映了R23侧链在酶的三元复合体中结合和定位辅因子的直接作用。R178突变使Dump的K-m增加40-2000倍，使辅因子的K-m增加3-20倍，对k(CAT)没有显著影响。这些结果与以下事实相一致，即该残基是转储结合壁的组成部分，并有助于其他几个转储结合残基的取向和有序化。除R179P外，所有R179突变的动力学参数与野生型值没有显著差异，反映了这种外部精氨酸不直接接触辅因子或其他配体结合残基的事实。R218对于催化位点的结构是必不可少的，除R218K外，该精氨酸的所有突变都是无效的。
Arginines R23, R178, R179 and R218 in thymidylate synthase (TS, EC 2.1.1.45) are hydrogen bond donors to the phosphate moiety of the substrate, dUMP, In order to investigate how these arginines contribute to enzyme function, we prepared complete replacement sets of mutants at each of the four sites in Lactobacillus casei TS, Mutations of R23 increase K-m for dUMP 2-20-fold, increase K-m for cofactor 8-40-fold and decrease k(cat) 9-20-fold, reflecting the direct role of the R23 side chain in binding and orienting the cofactor in ternary complexes of the enzyme. Mutations of R178 increase K-m for dUMP 40-2000-fold, increase K-m for cofactor 3-20-fold and do not significantly affect k(cat). These results are consistent with the fact that this residue is an integral part of the dUMP-binding wall and contributes to the orientation and ordering of several other dUMP binding residues. Kinetic parameters for all R179 mutations except R179P were not significantly different from wildtype values, reflecting the fact that this external arginine does not directly contact the cofactor or other ligand-binding residues. R218 is essential for the structure of tbe catalytic site and all mutations of this arginine except R218K were inactive.