title: Methanococcus jannaschii prolyl-cysteinyl-tRNA synthetase possesses overlapping amino acid binding sites.
abstract: 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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8114, USA.
authors.forename: C
authors.initials: C
authors.lastname: Stathopoulos
authors.forename: C
authors.initials: C
authors.lastname: Jacquin-Becker
authors.forename: H D
authors.initials: HD
authors.lastname: Becker
authors.forename: T
authors.initials: T
authors.lastname: Li
authors.forename: A
authors.initials: A
authors.lastname: Ambrogelly
authors.forename: R
authors.initials: R
authors.lastname: Longman
authors.forename: D
authors.initials: D
authors.lastname: Söll
mesh_terms: D000215:Acylation; D020816:Amino Acid Motifs / Q000235:genetics; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D007700:Kinetics; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D009097:Multienzyme Complexes; D012346:RNA, Transfer, Amino Acyl; D011392:Proline; D000604:Amino Acyl-tRNA Synthetases; C100134:prolyl T RNA synthetase; C019975:cysteinyl-tRNA synthetase; D003545:Cysteine