PMID:1712632
| Citation |
Miller, WT, Hill, KA and Schimmel, P (1991) Evidence for a "cysteine-histidine box" metal-binding site in an Escherichia coli aminoacyl-tRNA synthetase. Biochemistry 30:6970-6 |
|---|---|
| Abstract |
Escherichia coli alanyl-tRNA synthetase contains the sequence Cys-X2-Cys-X6-His-X2-His. This motif is distinct from the zinc fingers of DNA-binding proteins but has some similarity to the Cys-X2-Cys-X4-His-X4-Cys zinc-binding motif of retroviral gag proteins, where it has a role in RNA packaging. In Ala-tRNA synthetase, this sequence is located in an amino-terminal domain which has the site for docking the acceptor end of the tRNA near the bound aminoacyl adenylate and is immediately adjacent in the sequence to the location of a mutation that affects the specificity of tRNA recognition. We show here that Ala-tRNA synthetase contains approximately 1 mol of zinc/mol of polypeptide and that addition of the zinc chelator 1,10-phenanthroline inhibits its aminoacylation activity. Conservative mutations of specific cysteine or histidine residues in the "Cys-His box" destabilize and inactivate the enzyme, whereas mutations of intervening amino acids do not inactivate. The possibility that this motif can bind zinc (or cobalt) was demonstrated with a synthetic 22 amino acid peptide that is based on the sequence of the alanine enzyme. The peptide-cobalt complex has the spectral characteristics of tetrahedral coordination geometry. The results establish that the Cys-His box motif of Ala-tRNA synthetase has the potential to form a specific complex with zinc (at least in the context of a synthetic peptide analogue) and suggest that this motif is important for enzyme stability/activity. |
| Links | |
| Keywords |
Alanine-tRNA Ligase/chemistry; Alanine-tRNA Ligase/genetics; Amino Acid Sequence; Blotting, Western; Carrier Proteins/genetics; Cysteine/chemistry; Cysteine/genetics; Cysteine/metabolism; Escherichia coli/drug effects; Escherichia coli/enzymology; Escherichia coli/genetics; Histidine/chemistry; Histidine/genetics; Histidine/metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments/genetics; Peptide Fragments/isolation & purification; Peptide Fragments/metabolism; RNA/metabolism; Spectrophotometry, Atomic; Structure-Activity Relationship; Zinc/chemistry; Zinc/metabolism |
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