Identification of structurally and functionally important histidine residues in cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae.
Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is an alpha 2 dimer (alpha, Mr 63,000), each alpha containing 12 histidines. The covalent incorporation of 6-7 mol of diethyl pyrocarbonate per monomer corresponded to complete enzyme inactivation. This inactivation was reversed by hydroxylamine hydrolysis which regenerates free histidine (and tyrosine) while leaving the carbethoxy group still attached to the epsilon-amino group of lysine. Three histidines, one tyrosine, and four lysines were the main targets of the reagent. Site-directed mutagenesis was also tried to replace each of these modified residues. Given the unstability of the carbethoxy-imidazole bond, the nine histidines that were not modified by diethyl pyrocarbonate were mutated too. For these experiments, the enzyme was expressed in Escherichia coli by using a vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene. This substitution had no effect on the kinetic parameters. The combined results of chemical modification and site-directed mutagenesis show that one histidine seems to be part of the active site while two others play an important structural role. On the other hand, labeled lysines and tyrosine are nonessential residues. These results are discussed in light of two recent articles establishing the existence of a second family of aminoacyl-tRNA synthetases devoid of the HIGH and KMSKS consensus sequences and containing no Rossmann's domain in their three-dimensional structures.
Gasparini
S
S
Laboratoire de Biochimie, Centre National de la Recherche Scientifique, Strasbourg, France.
Vincendon
P
P
Eriani
G
G
Gangloff
J
J
Boulanger
Y
Y
Reinbolt
J
J
Kern
D
D
D000595:Amino Acid Sequence; D001226:Aspartate-tRNA Ligase / Q000235:genetics*; D006639:Histidine / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D006639:Histidine; D001226:Aspartate-tRNA Ligase