RESUMO
The tRNA-dependent amino acid activation catalyzed by mammalian arginyl-tRNA synthetase has been characterized. A conditional lethal mutant of Chinese hamster ovary cells that exhibits reduced arginyl-tRNA synthetase activity (Arg-1), and two of its derived revertants (Arg-1R4 and Arg-1R5) were analyzed at the structural and functional levels. A single nucleotide change, resulting in a Cys to Tyr substitution at position 599 of arginyl-tRNA synthetase, is responsible for the defective phenotype of the thermosensitive and arginine hyper-auxotroph Arg-1 cell line. The two revertants have a single additional mutation resulting in a Met222 to Ile change for Arg-1R4 or a Tyr506 to Ser change for Arg-1R5. The corresponding mutant enzymes were expressed in yeast and purified. The Cys599 to Tyr mutation affects both the thermal stability of arginyl-tRNA synthetase and the kinetic parameters for arginine in the ATP-PP(i) exchange and tRNA aminoacylation reactions. This mutation is located underneath the floor of the Rossmann fold catalytic domain characteristic of class 1 aminoacyl-tRNA synthetases, near the end of a long helix belonging to the alpha-helix bundle C-terminal domain distinctive of class 1a synthetases. For the Met222 to Ile revertant, there is very little effect of the mutation on the interaction of arginyl-tRNA synthetase with either of its substrates. However, this mutation increases the thermal stability of arginyl-tRNA synthetase, thereby leading to reversion of the thermosensitive phenotype by increasing the steady-state level of the enzyme in vivo. In contrast, for the Arg-1R5 cell line, reversion of the phenotype is due to an increased catalytic efficiency of the C599Y/Y506S double mutant as compared to the initial C599Y enzyme. In light of the location of the mutations in the 3D structure of the enzyme modeled using the crystal structure of the closely related yeast arginyl-tRNA synthetase, the kinetic analysis of these mutants suggests that the obligatory tRNA-induced activation of the catalytic site of arginyl-tRNA synthetase involves interdomain signal transduction via the long helices that build the tRNA-binding domain of the enzyme and link the site of interaction of the anticodon domain of tRNA to the floor of the active site.
Assuntos
Arginina-tRNA Ligase/química , Arginina-tRNA Ligase/metabolismo , Arginina/genética , Arginina/metabolismo , RNA de Transferência de Arginina/genética , RNA de Transferência de Arginina/metabolismo , Acilação , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Arginina-tRNA Ligase/genética , Arginina-tRNA Ligase/isolamento & purificação , Sítios de Ligação , Células CHO , Clonagem Molecular , Cricetinae , DNA Complementar/genética , Estabilidade Enzimática , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNA , Transdução de Sinais , Supressão Genética/genética , TermodinâmicaRESUMO
In mammalian cells, the nine aminoacyl-tRNA synthetases (aaRS) specific for the amino acids (aa) Glu, Pro, Ile, Leu, Met, Gln, Lys, Arg and Asp are associated within a multienzyme complex. Arginyl-tRNA synthetase (ArgRS) is characterized by the occurrence of two structurally distinct forms of that enzyme: a complexed (approximately 74 kDa) and a free (approximately 60 kDa) form. The cDNA encoding the 74-kDa species of ArgRS from Chinese hamster ovary cells has been isolated and sequenced. The deduced aa sequence shows 38% identity to the homologous bacterial enzyme but displays an N-terminal polypeptide extension composed of 73 aa, which is absent in the free form of mammalian ArgRS. Two regions of this extension are predicted to be alpha-helical, leading to the clustering of Leu and Ile residues on one side of the helices. This suggests that the N-terminal domain is involved in the assembly of the 74-kDa species of ArgRS within the multisynthetase complex through hydrophobic interactions. By using the isolated cDNA, a Northern blot analysis showed a single mRNA species. Thus, there is a possibility that the free and complexed forms of ArgRS are encoded by the same gene.
Assuntos
Arginina-tRNA Ligase/genética , Complexos Multienzimáticos/genética , Sequência de Aminoácidos , Animais , Arginina-tRNA Ligase/química , Arginina-tRNA Ligase/metabolismo , Sequência de Bases , Northern Blotting , Células CHO , Clonagem Molecular , Cricetinae , DNA , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Estrutura Secundária de Proteína , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Água/químicaRESUMO
Native isoleucyl-tRNA synthetase and a structurally modified form of methionyl-tRNA synthetase were purified to homogeneity following trypsinolysis of the high molecular weight complex from sheep liver containing eight aminoacyl-tRNA synthetases. The correspondence between purified isoleucyl-tRNA synthetase and the previously unassigned polypeptide component of Mr 139 000 was established. It is shown that dissociation of this enzyme from the complex has no discernible effect on its kinetic parameters. Both isoleucyl- and methionyl-tRNA synthetases contain one zinc ion per polypeptide chain. In both cases, removal of the metal ion by chelating agents leads to an inactive apoenzyme. As the trypsin-modified methionyl-tRNA synthetase has lost the ability to associate with other components of the complex [Mirande, M., Kellermann, O., & Waller, J. P. (1982) J. Biol. Chem. 257, 11049-11055], the zinc ion is unlikely to be involved in complex formation. While native purified isoleucyl-tRNA synthetase displays hydrophobic properties, trypsin-modified methionyl-tRNA synthetase does not. It is suggested that the assembly of the amino-acyl-tRNA synthetase complex is mediated by hydrophobic domains present in these enzymes.