Study of the arrangement of the functional domains along the yeast cytoplasmic aspartyl-tRNA synthetase.
Aspartyl-tRNA synthetase from yeast (AspRS) was screened for functional domains by measuring the effect of two types of amino acid mutations on its catalytic properties: (a) insertion of a dipeptide or a tetrapeptide along the polypeptide chain, (b) deletion of various lengths from the enzyme C-terminal. It was shown that insertion mutations significantly affect the kinetic properties of AspRS only when occurring in the second quarter of the molecule and the two centrally located mutations even inactivate the enzyme completely. Analysis of kinetic data strongly suggests that, in fact, all the observed activity modifications result from alteration of the activation reaction rate constant, kappa cat only. This led to the conclusion that the domain involved in aspartic acid activation should be located in the second quarter of the molecule. Furthermore, a deletion mutant with a modification of the last five amino acid residues was isolated. This mutant is fully active in the activation step, but has lost 80% of the wild-type aminoacylation activity. This involvement of the C-terminus in acylation implies that it has to be folded towards strategic regions of the enzyme, thus favouring conformations required for catalysis or maintaining the tRNA in a functional position.
Prevost
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G
Institut de Biologie Moléculaire et Cellulaire du CNRS et Université Louis Pasteur, Strasbourg, France.
Eriani
G
G
Kern
D
D
Dirheimer
G
G
Gangloff
J
J
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D001226:Aspartate-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005800:Genes, Fungal; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D003062:Codon; D000604:Amino Acyl-tRNA Synthetases; D001226:Aspartate-tRNA Ligase