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.
Highlights
During the first of two partial reactions in aminoacylation, the cognate amino acid is condensed with ATP to form an aminoacyl-adenylate
Comparison of the kinetics of substrate-induced fluorescence changes to the kinetics of product formation determined by rapid quench suggests that adenylation rates are asymmetric with respect to the two active sites of the dimer, and that conformational changes linked to the insertion domain may be rate-limiting for product formation
Design of a Modified histidyl-tRNA synthetase (HisRS) Readily Labeled with Fluorescence Probes and Equilibrium Fluorescence Measurements—To investigate the conformational changes within the enzyme active site, an engineered variant of HisRS was generated by site-directed mutagenesis that could be labeled with an extrinsic environmentally sensitive fluorophore at a unique location (Fig. 1A)
Summary
During the first of two partial reactions in aminoacylation, the cognate amino acid is condensed with ATP to form an aminoacyl-adenylate. A detailed pre-steady-state analysis of mutants of tRNAHis or HisRS compromised with respect to tRNA identity suggested that, in the complete aminoacylation reaction, formation of aminoacyl adenylate in the second active site is contingent upon a productive aminoacyl transfer reaction in the first [20] These and other data led to the proposal of an alternating site model for HisRS [20] that is analogous to the “flip flop” catalysis suggested for class II PheRS [21, 22] and class Ic TrpRS [14]. Comparison of the kinetics of substrate-induced fluorescence changes to the kinetics of product formation determined by rapid quench suggests that adenylation rates are asymmetric with respect to the two active sites of the dimer, and that conformational changes linked to the insertion domain may be rate-limiting for product formation The implications of these results for a previous model [20] of alternating site catalysis in HisRS are discussed
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