PMID:7488160
| Citation |
Zhou, L and Rosevear, PR (1995) Mutation of the carboxy terminal zinc finger of E. coli isoleucyl-tRNA synthetase alters zinc binding and aminoacylation activity. Biochem. Biophys. Res. Commun. 216:648-54 |
|---|---|
| Abstract |
Escherichia coli isoleucyl-tRNA synthetase has been shown to contain two enzyme-bound zinc atoms per polypeptide chain. To investigate the structural and functional significance of the C-terminal enzyme-bound zinc, mutagenesis was used to alter Cys 922 to Ser [IleRS(C922S)] and to replace Cys 922 through Ala 939 with a 33 amino acid peptide unable to bind zinc (AIleRS). Both IleRS(C922S) and AIleRS were found to contain only a single enzyme-bound zinc per polypeptide chain. Substitution of Co2+ for Zn2+ in IleRS(C922S) gave a visible spectrum characteristic of that expected for a single tetrahedrally coordinated enzyme-bound Co2+ atom. Little or no effect on the Km values for ATP or Ile and only a 5 fold reduction of the (kcat/Km)Ile was observed for IleRS(C922S) and AIleRS in the isoleucine-dependent ATP-pyrophosphate exchange reaction. In the tRNA-dependent aminoacylation reaction, Km values for tRNA(Ile) were only slightly affected in the mutant proteins. However, kcat/Km values were decreased approximately 200 and 2500 fold for IleRS(C922S) and AIleRS, respectively. These results suggest that both the C-terminal enzyme-bound zinc and the C-terminal peptide play important roles in aminoacylation of tRNA(Ile). |
| Links |
PubMed Online version:10.1006/bbrc.1995.2671 |
| Keywords |
Amino Acid Sequence; Base Sequence; Cysteine; DNA Primers; Enterobacter/enzymology; Escherichia coli/enzymology; Escherichia coli/genetics; Isoleucine-tRNA Ligase/genetics; Isoleucine-tRNA Ligase/metabolism; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Point Mutation; Polymerase Chain Reaction; Pseudomonas fluorescens/enzymology; Recombinant Proteins/metabolism; Sequence Homology, Amino Acid; Serine; Staphylococcus aureus/enzymology; Zinc Fingers |
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