NDLI: Asymmetric Amino Acid Activation by Class II Histidyl-tRNA Synthetase from Escherichia coli *

Content Provider	PubMed Central
Author	Guth, Ethan Farris, Mindy Bovee, Michael Francklyn, Christopher S.
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.
Related Links	http://dx.doi.org/10.1074/jbc.m109.021311
Ending Page	20762
Page Count	10
Starting Page	20753
File Format	PDF
ISSN	00219258
e-ISSN	1083351X
Journal	The Journal of Biological Chemistry
Issue Number	31
Volume Number	284
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2009-07-31
Access Restriction	Open
Rights Holder	American Society for Biochemistry and Molecular Biology
Subject Keyword	Cell Biology Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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Asymmetric Amino Acid Activation by Class II Histidyl-tRNA Synthetase from Escherichia coli

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