假单胞菌YZ-26来源环酰亚胺水解酶中半胱氨酸残基的反应性及功能

Reactivity and function of cysteine residues in imidase from Pseudomonas putida YZ-26

【目的】研究环酰亚胺水解酶(Imidase,CIH)中的两个半胱氨酸残基的反应性及功能。【方法】设计了3个半胱氨酸突变酶:CIH7,108、CIH7、CIH108。将天然酶以及突变酶基因分别与麦芽糖结合蛋白(MBP)基因在大肠杆菌(Escherichia coli)中进行融合表达,融合蛋白经纯化后得到了电泳纯的样品。使用5,5'-二硫代双(2-硝基苯甲酸)(DTNB)对天然酶CIH的巯基基团进行修饰,并分析了DTT对分子状态的影响。进一步研究了经H2O2处理后CIH及其突变酶的锌离子结合能力及分子状态。【结果】酶活测定表明CIH7,108和CIH7的活力基本丧失,而CIH108仍保持了72%的酶活性。CIH中的两个半胱氨酸残基以游离形式存在,不形成链内或链间二硫键。CIH与CIH108为四聚体结构且具有一定的锌离子结合能力,CIH7,108为多聚体,CIH7为单体及多体的混合物且都不具备锌离子结合能力,随着H2O2浓度的增加,CIH中的链内二硫键及CIH108中的链间二硫键逐渐增加。【结论】说明Cys7是结合锌离子和稳定CIH分子结构的必要残基。

[Objective] We investigated the reactivity and function of two cysteine residues in imidase（CIH） by site-directed mutagenesis.[Methods] Three variants of imidase（CIH） were constructed with Cys7 and Cys108 or only one of them substituted with Gly.The two thiol groups of Cys7 and Cys108 of imidase were specifically modified separately or collectively by dithio-bis-nitrobenzoic acid（DTNB） in the native state.It was also confirmed by SDS-PAGE analysis.To further verify the above results,the oligomeric structure and sulfhedryl groups of native and mutant CIH were also examined by measuring zinc binding ability and molecular size under different concentrations of H2O2.[Results] Compared with CIH,CIH108 retained 72% activity,while CIH7,108 and CIH7 had no activity using DL-hydantoin as substrate.The spectral detection result shown that the two thiol groups were both in a free state.It is indicated that CIH and CIH108 are tetramer,CIH7,108 is multimer,and CIH7 is a mixture of monomer and multimer.The zinc binding ability of CIH108 was still relative high,while CIH7 and CIH7,108 decreased obviously.The increased concentration of H2O2 could increase the intrachain disulfide bond of CIH and the interchain disulfide bond of CIH108.[Conclusion] All data imply that the Cys7 is required for binding zinc ion and maintaining the stable structure of enzymatic molecule.