Research article summary (published 30 May 2009):

Asymmetric amino acid activation by class II histidyl-tRNA synthetase from Escherichia coli.

Full Abstract

Aminoacyl-tRNA synthetases (ARSs) join amino acids to their cognate tRNAs to initiate protein synthesis. Class II ARS possess a unique catalytic domain fold, possess active site signature sequences, and are dimers or tetramers. The dimeric class I enzymes, notably TyrRS, exhibit half-of-sites reactivity, but its mechanistic basis is unclear. In class II histidyl-tRNA synthetase (HisRS), amino acid activation occurs at different rates in the two active sites when tRNA is absent, but half-of-sites reactivity has not been observed. To investigate the mechanistic basis of the asymmetry, and explore the relationship between adenylate formation and conformational events in HisRS, a fluorescently labeled version of the enzyme was developed by conjugating 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) to a cysteine introduced at residue 212, located in the insertion domain. The binding of the substrates histidine, ATP, and 5'-O-[N-(l-histidyl)sulfamoyl]adenosine to MDCC-HisRS produced fluorescence quenches on the order of 6-15%, allowing equilibrium dissociation constants to be measured. The rates of adenylate formation measured by rapid quench and domain closure as measured by stopped-flow fluorescence were similar and asymmetric with respect to the two active sites of the dimer, indicating that conformational change may be rate-limiting for product formation. Fluorescence resonance energy transfer experiments employing differential labeling of the two monomers in the dimer suggested that rigid body rotation of the insertion domain accompanies adenylate formation. The results support an alternating site model for catalysis in HisRS that may prove to be common to other class II aminoacyl-tRNA synthetases.

Author information

Author/s: Guth, Ethan (E); Farris, Mindy (M); Bovee, Michael (M); Francklyn, Christopher S (CS);

Affiliation: Department of Biochemistry, College of Medicine, Health Sciences Complex, University of Vermont, Burlington, Vermont 05405, USA.

Grants: F32 GM19739 (Agency:NIGMS NIH HHS) ; R01 GM54899 (Agency:NIGMS NIH HHS)

Journal and publication information

Publication Type: Journal Article; Research Support, N.I.H., Extramural

Journal: The Journal of biological chemistry (J Biol Chem), published in United States. (Language: eng)

Reference: 2009-Jul; vol 284 (issue 31) : pp 20753-62

Dates: Created 2009/07/27; Completed 2009/10/14;

PMID: 19487703, status: MEDLINE (last retrieved date: 10/14/2009)

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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MeSH headings (categories)

This article was linked to the MeSH Headings (categories) shown below.

Adenosine Monophosphate - metabolism Adenosine Triphosphate - metabolism Aminoacylation Energy Transfer Escherichia coli - enzymology Fluorescent Dyes - metabolism Histidine-tRNA Ligase - chemistry; metabolism

Hydrogen-Ion Concentration Kinetics Protein Multimerization Protein Structure, Secondary Spectrometry, Fluorescence Substrate Specificity Temperature

Note: Bold headings indicate primary MeSH headings or qualifiers.

Associated Chemicals: Fluorescent Dyes (0) ; Adenosine Triphosphate (56-65-5) ; Adenosine Monophosphate (61-19-8) ; Histidine-tRNA Ligase (EC 6.1.1.21)

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