Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae. Study of its functional organisation by deletion analysis.
Aspartyl-tRNA synthetase (AspRS) from yeast, a homodimer of 125 kDa, was shortened by several residues from the C- and N-termini, via site-directed mutagenesis, to examine the contribution of the removed peptides to the enzyme properties. This study showed that the N-terminal sequence up to amino acid 70 (which confers peculiar ionic properties to the protein) is dispensable for activity. Domains located beyond amino acid 70 appeared to have increasing catalytic importance; the removal of 80 or 90 residues affected the Km values for ATP and deletions of 101 or 140 amino acids profoundly modified the physiochemical properties of AspRS, and by consequence, its structural organisation (extraction of the mutated proteins out of the cells required the presence of SDS). On the C-terminal side, very limited modifications readily affected the enzyme properties. Deletion of as few as three residues increased the Km for ATP and reduced the aminoacylation kcat as well as the thermostability of the adenylate synthesis activity; the kcat of this step was impaired after deletion of two further residues. Finally, shortening the C-terminal decapeptide completely inactivated AspRS, whilst affecting neither its affinity for tRNAAsp nor its dimerisation capacity. These data reveal the role of the C-terminal decapeptide as a determinant in both reactions catalysed by AspRS. This peptide is involved in ATP binding, stabilising the functional conformation of the amino-acid-activating domain and probably maintaining the tRNA-acceptor end in a reactive position with regard to the activated amino acid.
Eriani
G
G
Institut de Biologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Strasbourg, France.
Prevost
G
G
Kern
D
D
Vincendon
P
P
Dirheimer
G
G
Gangloff
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001226:Aspartate-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002872:Chromosome Deletion; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000255:Adenosine Triphosphate; D004247:DNA; D001226:Aspartate-tRNA Ligase