PMID:7797500
| Citation |
Liu, J, Gagnon, Y, Gauthier, J, Furenlid, L, L'Heureux, PJ, Auger, M, Nureki, O, Yokoyama, S and Lapointe, J (1995) The zinc-binding site of Escherichia coli glutamyl-tRNA synthetase is located in the acceptor-binding domain. Studies by extended x-ray absorption fine structure, molecular modeling, and site-directed mutagenesis. J. Biol. Chem. 270:15162-9 |
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| Abstract |
The zinc contents of fragments of Escherichia coli glutamyl-tRNA synthetase, as well as the conservation of the CYC sequence only in zinc-containing glutamyl-tRNA synthetases, suggested that the 98CYCX24-CRHSHEHHADDEPC138 includes some or all residues involved in binding its zinc atom (Liu, J., Lin, S.-X., Blochet, J.-E., Pézolet, M., and Lapointe, J. (1993) Biochemistry 32, 11390-11396). Extended x-ray absorption fine structure (EXAFS) shows that this zinc atom has a four-coordinate non-planar coordination environment with 3 sulfur and 1 nitrogen atoms with bond lengths, respectively, 2.37 +/- 0.02 A and 2.01 +/- 0.02 A, presumably belonging to 3 cysteine residues and 1 histidine residue. Conservative replacement of each histidine and cysteine residue of the 98C-138C segment, respectively, with glutamine (Q) and serine (S), yields variants H129Q, H131Q, H132Q, and C138S (which sustain the growth at 42 degrees C of E. coli JP1449, whose glutamyl-tRNA synthetase is thermosensitive) and C98S, C100S, C125S, and H127Q (which do not). The amount of this enzyme in these mutants is at least 1 order of magnitude larger than that in a wild type strain; however, no glutamyl-tRNA synthetase activity is detectable in extracts of the variants C100S and C125S, whereas its specific activity in those of C98S and H127Q is about 10-fold lower than in cells overproducing the wild type enzyme or the variants H129Q, H131Q, H132Q, and C138S. These results indicate that the zinc atom present in E. coli glutamyl-tRNA synthetase is bound by the 2 evolutionarily conserved cysteines at positions 98 and 100, and by Cys125 and His127. Molecular modeling of the N-terminal half of this enzyme, using the known structure of E. coli glutaminyl-tRNA synthetase, supports this conclusion and suggests that the 98C-127H segment does not have the characteristics of the classical zinc fingers. |
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| Keywords |
Absorptiometry, Photon/methods; Amino Acid Sequence; Animals; Bacteria/enzymology; Binding Sites; Drosophila melanogaster/enzymology; Escherichia coli/enzymology; Escherichia coli/genetics; Glutamate-tRNA Ligase/biosynthesis; Glutamate-tRNA Ligase/chemistry; Glutamate-tRNA Ligase/metabolism; Humans; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Protein Structure, Secondary; Recombinant Proteins/biosynthesis; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Sequence Homology, Amino Acid; Zinc/metabolism |
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