Mapping of the zinc binding domain of Escherichia coli methionyl-tRNA synthetase.
Cys/His motifs, found in several nucleic acid binding proteins, generally correspond to sites for the binding of metal atoms. Such a motif, comprising four Cys residues, occurs in the subunits of Escherichia coli methionyl-tRNA synthetase, a dimeric enzyme known to bind two zinc atoms. In this study, each of the four cysteines in the cysteine cluster (region 145 to 161) of E. coli methionyl-tRNA synthetase were successively changed into an alanine. Either substitution is sufficient to destabilize the tight binding of the zinc ion. Moreover, a peptide having a sequence corresponding to that of the 138 to 163 region of methionyl-tRNA synthetase has been prepared. It strongly binds one zinc atom, even in the presence of ethylene diamine tetraacetate. These data establish that, in methionyl-tRNA synthetase, the Cys motif of region 145 to 161 is actually the binding site for zinc. In addition, the mutation of each cysteine modifies the parameters of the methionine activation reaction, and appears to change the structure of the enzyme, as probed by an increased sensitivity of the mutant enzymes to trypsin attack. The possible role of the zinc atom and of its chelating residues in the folding of the active centre of methionyl-tRNA synthetase is discussed.
Fourmy
D
D
Unité de Recherche Associée 240 du Centre National de la Recherche Scientifique Ecole Polytechnique, Palaiseau, France.
Meinnel
T
T
Mechulam
Y
Y
Blanquet
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D000412:Alanine-tRNA Ligase / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology*; D008718:Methionine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013054:Spectrophotometry, Atomic; D014357:Trypsin / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D014357:Trypsin; D008718:Methionine-tRNA Ligase; D000412:Alanine-tRNA Ligase; D015032:Zinc; D003545:Cysteine