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

C. Stathopoulos, C. Jacquin-Becker, H. D. Becker, T. Li, A. Ambrogelly, R. Longman, D. Söll

Research output: Contribution to journalArticle

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-PP1 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 tRNACys promotes cysteine activation. The kcat 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.

Original languageEnglish (US)
Pages (from-to)46-52
Number of pages7
JournalBiochemistry
Volume40
Issue number1
DOIs
StatePublished - Jan 9 2001
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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    Stathopoulos, C., Jacquin-Becker, C., Becker, H. D., Li, T., Ambrogelly, A., Longman, R., & Söll, D. (2001). Methanococcus jannaschii prolyl-cysteinyl-tRNA synthetase possesses overlapping amino acid binding sites. Biochemistry, 40(1), 46-52. https://doi.org/10.1021/bi002108x