Methanococcus jannaschii prolyl-cysteinyl-tRNA synthetase possesses overlapping amino acid binding sites.

TitleMethanococcus jannaschii prolyl-cysteinyl-tRNA synthetase possesses overlapping amino acid binding sites.
Publication TypeJournal Article
Year of Publication2001
AuthorsStathopoulos C, Jacquin-Becker C, Becker HD, Li T, Ambrogelly A, Longman R, Söll D
Date Published2001 Jan 9
KeywordsAcylation, Amino Acid Motifs, Amino Acyl-tRNA Synthetases, Animals, Archaeal Proteins, Binding Sites, Cysteine, Enzyme Activation, Humans, Kinetics, Methanococcus, Multienzyme Complexes, Mutagenesis, Site-Directed, Proline, RNA, Transfer, Amino Acyl

The protein translation apparatus of Methanococcus jannaschii possesses the unusual enzyme prolyl-cysteinyl-tRNA synthetase (ProCysRS), a single enzyme that attaches two different amino acids, proline and cysteine, to their cognate tRNA species. Measurement of the ATP-PP(i) exchange reaction revealed that amino acid activation, the first reaction step, differs for the two amino acids. While Pro-AMP can be formed in the absence of tRNA, Cys-AMP synthesis is tRNA-dependent. Studies with purified tRNAs indicate that tRNA(Cys) promotes cysteine activation. The k(cat) values of wild-type ProCysRS for tRNA prolylation (0.09 s(-1)) and cysteinylation (0.02 s(-1)) demonstrate that both aminoacyl-tRNAs are synthesized with comparable rates, the cysteinyl-tRNA synthetase activity being only 4.5-fold lower than prolyl-tRNA synthetase activity. Kinetic analysis of ProCysRS mutant enzymes, generated by site-directed mutagenesis, shows glutamate at position 103 to be critical for proline binding, and proline at position 100 to be involved in cysteine binding. The proximity in ProCysRS of amino acid residues affecting binding of either cysteine or proline strongly suggests that structural elements of the two amino acid binding sites overlap.

Alternate JournalBiochemistry
PubMed ID11141055