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Substrate and Enzyme Functional Groups Contribute to Translational Quality Control by Bacterial Prolyl-tRNA Synthetase
journal contribution
posted on 2012-06-14, 00:00 authored by Sandeep Kumar, Mom Das, Christopher M. Hadad, Karin Musier-ForsythAminoacyl-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 tRNAPro. Editing of
the misacylated aminoacyl-tRNA is required for error-free protein
synthesis. An editing domain (INS) appended to bacterial ProRS selectively
hydrolyzes Ala-tRNAPro, whereas Cys-tRNAPro 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-tRNAPro 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.
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Keywords
chain atomsediting domainExperimental studiesfreestanding editing domainOHTranslational Quality Controltransition state stabilizationtRNA moleculesGXXXP loopINSresults supportcatalysiwater activationsubstrate bindingsynthetaseprotein synthesisEnzyme Functional Groupsester linkageGly 261mechanismrolesubstrate sulfhydryl chemistryalanine specificitymisacylate tRNAProacid substratesProRSQM
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