Mutation of the carboxy terminal zinc finger of E. coli isoleucyl-tRNA synthetase alters zinc binding and aminoacylation activity.
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).
D000595:Amino Acid Sequence; D001483:Base Sequence; D003545:Cysteine; D017931:DNA Primers; D004754:Enterobacter / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007533:Isoleucine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011551:Pseudomonas fluorescens / Q000201:enzymology; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine; D013211:Staphylococcus aureus / Q000201:enzymology; D016335:Zinc Fingers
D017931:DNA Primers; D011994:Recombinant Proteins; D012694:Serine; D007533:Isoleucine-tRNA Ligase; D003545:Cysteine