Effect of cysteine residues on the activity of arginyl-tRNA synthetase from Escherichia coli.
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.
Liu
M
M
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Academia Sinica, China.
Huang
Y
Y
Wu
J
J
Wang
E
E
Wang
Y
Y
D000215:Acylation; D001126:Arginine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005033:Ethylmaleimide / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D007460:Iodoacetamide / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013439:Sulfhydryl Reagents / Q000737:chemistry; D017186:Titrimetry
D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D001126:Arginine-tRNA Ligase; D003545:Cysteine; D005033:Ethylmaleimide; D007460:Iodoacetamide