Liu M, Huang Y, Wu J, Wang E, Wang Y
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Academia Sinica, China.
Biochemistry. 1999 Aug 24;38(34):11006-11. doi: 10.1021/bi990392q.
Arginyl-tRNA synthetase (ArgRS) from Escherichia coli (E. coli) contains four cysteine residues. In this study, the role of cysteine residues in the enzyme has been investigated by chemical modification and site-directed mutagenesis. Titration of sulfhydryl groups in ArgRS by 5, 5'-dithiobis(2-nitro benzoic acid) (DTNB) suggested that a disulfide bond was not formed in the enzyme and that, in the native condition, two DTNB-sensitive cysteine residues were located on the surface of ArgRS, while the other two were buried inside. Chemical modification of the native enzyme by iodoacetamide (IAA) affected only one DTNB-sensitive cysteine residue and resulted in 50% loss of enzyme activity, while modification by N-ethylmeimide (NEM) affected two DTNB-sensitive residues and caused a complete loss of activity. These results, when combined with substrate protection experiments, suggested that at least the two cysteine residues located on the surface of the molecule were directly involved in substrates binding and catalysis. However, changing Cys to Ala only resulted in slight loss of enzymatic activity and substrate binding, suggesting that these four cysteine residues in E. coli ArgRS were not essential to the enzymatic activity. Moreover, modifications of the mutant enzymes indicated that the two DTNB- and NEM-sensitive residues were Cys(320) and Cys(537) and the IAA-sensitive was Cys(320). Our study suggested that inactivation of E. coli ArgRS by sulfhydryl reagents is a result of steric hindrance in the enzyme.
来自大肠杆菌（E. coli）的精氨酰 - tRNA合成酶（ArgRS）含有四个半胱氨酸残基。在本研究中，通过化学修饰和定点诱变研究了半胱氨酸残基在该酶中的作用。用5,5'-二硫代双（2 - 硝基苯甲酸）（DTNB）滴定ArgRS中的巯基表明，该酶中未形成二硫键，并且在天然状态下，两个对DTNB敏感的半胱氨酸残基位于ArgRS的表面，而另外两个则埋藏在内部。用碘乙酰胺（IAA）对天然酶进行化学修饰仅影响一个对DTNB敏感的半胱氨酸残基，并导致酶活性丧失50％，而用N - 乙基马来酰亚胺（NEM）修饰则影响两个对DTNB敏感的残基并导致活性完全丧失。这些结果与底物保护实验相结合表明，至少位于分子表面的两个半胱氨酸残基直接参与底物结合和催化。然而，将半胱氨酸突变为丙氨酸仅导致酶活性和底物结合略有丧失，这表明大肠杆菌ArgRS中的这四个半胱氨酸残基对酶活性并非必不可少。此外，对突变酶的修饰表明，两个对DTNB和NEM敏感的残基是Cys(320)和Cys(537)，而对IAA敏感的是Cys(320)。我们的研究表明，巯基试剂使大肠杆菌ArgRS失活是酶中空间位阻的结果。
