title: Substrate and enzyme functional groups contribute to translational quality control by bacterial prolyl-tRNA synthetase.
abstract: Aminoacyl-tRNA synthetases activate specific amino acid substrates and attach them via an ester linkage to cognate tRNA molecules. In addition to cognate proline, prolyl-tRNA synthetase (ProRS) can activate cysteine and alanine and misacylate tRNA(Pro). Editing of the misacylated aminoacyl-tRNA is required for error-free protein synthesis. An editing domain (INS) appended to bacterial ProRS selectively hydrolyzes Ala-tRNA(Pro), whereas Cys-tRNA(Pro) is cleared by a freestanding editing domain, YbaK, through a unique mechanism involving substrate sulfhydryl chemistry. The detailed mechanism of catalysis by INS is currently unknown. To understand the alanine specificity and mechanism of catalysis by INS, we have explored several possible mechanisms of Ala-tRNA(Pro) deacylation via hybrid QM/MM calculations. Experimental studies were also performed to test the role of several residues in the INS active site as well as various substrate functional groups in catalysis. Our results support a critical role for the tRNA 2'-OH group in substrate binding and catalytic water activation. A role is also proposed for the protein's conserved GXXXP loop in transition state stabilization and for the main chain atoms of Gly261 in a proton relay that contributes substantially to catalysis.
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Kumar
authors.forename: Mom
authors.initials: M
authors.lastname: Das
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Hadad
authors.forename: Karin
authors.initials: K
authors.lastname: Musier-Forsyth
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013293:Enterococcus faecalis / Q000201:enzymology*; D008958:Models, Molecular; D011786:Quality Control; D011789:Quantum Theory; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D000604:Amino Acyl-tRNA Synthetases