Mutation of the carboxy terminal zinc finger of E. coli isoleucyl-tRNA synthetase alters zinc binding and aminoacylation activity

Biochem Biophys Res Commun. 1995 Nov 13;216(2):648-54. doi: 10.1006/bbrc.1995.2671.

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).

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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*

Substances

  • DNA Primers
  • Recombinant Proteins
  • Serine
  • Isoleucine-tRNA Ligase
  • Cysteine