Nonconserved residues Ala287 and Ser290 of the Cryptosporidium hominis thymidylate synthase domain facilitate its rapid rate of catalysis
Nonconserved residues Ala287 and Ser290 of the Cryptosporidium hominis thymidylate synthase domain facilitate its rapid rate of catalysis
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DOI：
10.1021/bi700531r
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发表时间：
2007-07-17
期刊：
影响因子：
2.9
通讯作者：
Anderson, Karen S.
中科院分区：
文献类型：
--
作者：
Doan, Lanxuan T.;Martucci, W. Edward;Anderson, Karen S.
人隐孢子虫TS-DHFR在TS结构域表现出异常高的催化速率，比其他TS酶的催化速率高至少10倍。使用定点诱变，我们突变的残基Ala 287和Ser290中的叶酸结合螺旋苯丙氨酸和甘氨酸，分别在人类和大多数其他TS酶的相应残基。我们的研究结果表明，突变体A287 F，突变体S290 G，和双突变体都有降低的亲和力亚甲基四氢叶酸和降低的TS结构域的反应速率。有趣的是，S290G突变体酶具有最低的TS活性，其催化效率比野生型（WT）低200倍。S290G突变体的构象变化速率比WT慢80倍，导致从氢化物转移到共价三元复合物形成的限速步骤发生变化。我们已经确定了配体结合的S290G突变酶的晶体结构，这表明突变的主要影响是TS配体之间的距离增加。动力学和晶体结构的数据提供了第一个证据，解释不寻常的快速TS速率在C。人类
Cryptosporidium hominis TS-DHFR exhibits an unusually high rate of catalysis at the TS domain, at least 10-fold greater than those of other TS enzymes. Using site-directed mutagenesis, we have mutated residues Ala287 and Ser290 in the folate-binding helix to phenylalanine and glycine, respectively, the corresponding residues in human and most other TS enzymes. Our results show that the mutant A287F, the mutant S290G, and the double mutant all have reduced affinities for methylene tetrahydrofolate and reduced rates of reaction at the TS domain. Interestingly, the S290G mutant enzyme had the lowest TS activity, with a catalytic efficiency similar to 200-fold lower than that of the wild type (WT). The rate of conformational change of the S290G mutant is similar to 80 times slower than that of WT, resulting in a change in the rate-limiting step from hydride transfer to covalent ternary complex formation. We have determined the crystal structure of ligand-bound S290G mutant enzyme, which shows that the primary effect of the mutation is an increase in the distance between the TS ligands. The kinetic and crystal structure data presented here provide the first evidence explaining the unusually fast TS rate in C. hominis.