González J C, Peariso K, Penner-Hahn J E, Matthews R G
Biophysics Research Division, University of Michigan, Ann Arbor 48109-1055, USA.
Biochemistry. 1996 Sep 24;35(38):12228-34. doi: 10.1021/bi9615452.
Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine. Previous work had shown the existence of a reactive thiol group, cysteine 726, whose alkylation led to loss of all detectable enzymatic activity [González, J.C., et al. (1992) Biochemistry 31, 6045-6056]. A site-directed mutation of MetE, Cys726Ser, was constructed to investigate the possible role of this cysteine. The Cys726Ser protein was purified to homogeneity, affording a protein with no detectable activity. To assess the possibility that cysteine726 functions as a metal ligand, inductively coupled plasma-atomic emission spectrometry was performed. The wild-type enzyme contains 1.02 equiv of zinc per subunit; the Cys726Ser mutant does not contain zinc, supporting the view that cysteine726 is required for metal binding. A loss of enzymatic activity is observed upon removal of zinc from the wild-type MetE by incubation in urea and EDTA; activity can subsequently be restored by zinc reconstitution, suggesting that zinc is required for catalysis. Circular dichroism measurements further suggest that there are no major differences in the secondary structures of the wild-type and the Cys726Ser mutant enzymes. Extended X-ray absorption fine structure analysis has established that the average zinc environment is different in the presence of homocysteine than in its absence and is consistent with the changes expected for displacement of an oxygen or nitrogen ligand by the sulfur of homocysteine. A possible model for zinc-dependent activation of homocysteine by MetE is presented.
来自大肠杆菌的钴胺素非依赖性蛋氨酸合酶（MetE）催化甲基从甲基四氢叶酸转移至同型半胱氨酸。先前的研究表明存在一个反应性巯基，即半胱氨酸726，其烷基化会导致所有可检测到的酶活性丧失[冈萨雷斯，J.C.等人（1992年）《生物化学》31卷，6045 - 6056页]。构建了MetE的一个定点突变体Cys726Ser，以研究该半胱氨酸可能发挥的作用。Cys726Ser蛋白被纯化至同质，得到一种无可检测活性的蛋白。为评估半胱氨酸726作为金属配体发挥作用的可能性，进行了电感耦合等离子体原子发射光谱分析。野生型酶每个亚基含有1.02当量的锌；Cys726Ser突变体不含锌，支持了半胱氨酸726是金属结合所必需的这一观点。通过在尿素和乙二胺四乙酸中孵育从野生型MetE中去除锌后，观察到酶活性丧失；随后通过锌重构可恢复活性，这表明催化作用需要锌。圆二色性测量进一步表明，野生型和Cys726Ser突变体酶的二级结构没有重大差异。扩展X射线吸收精细结构分析已确定，同型半胱氨酸存在时与不存在时锌的平均环境不同，且与同型半胱氨酸的硫取代氧或氮配体所预期的变化一致。本文提出了一个MetE对同型半胱氨酸进行锌依赖性激活的可能模型。
