Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases (original) (raw)
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Computational Insights into the High-Fidelily Catalysis of Aminoacyl-tRNA Synthetases
2017
Obtaining insights into the catalytic function of enzymes is an important area of research due to their widespread applications in the biotechnology and pharmaceutical industries. Among these enzymes, the aminoacyl-tRNA synthetases (aaRSs) are known for their remarkable fidelity in catalyzing the aminoacylation reactions of tRNA in protein biosynthesis. Despite the exceptional execution of this critical function, mechanistic details of the reactions catalyzed by aminoacyl-tRNA synthetases remain elusive demonstrating the obvious need to explore their remarkable chemistry. During the PhD studies reported in this thesis the mechanism of aminoacylation, pre-transfer editing and post-transfer editing catalyzed by different aaRS have been established using multi-scale computational enzymology. In the first two chapters a detailed information about aaRS and the addressed questions was given in addition to an overview of the used computational methodology currently used to investigate the ...
Principles of tRNAAla Selection by Alanyl–tRNA Synthetase Based on the Critical G3·U70 Base Pair
ACS Omega, 2019
Throughout evolution, the presence of a single G3•U70 mismatch in the acceptor stem of tRNA Ala is the major determinant for aminoacylation with alanine by alanyl−tRNA synthetase (AlaRS). Recently reported crystal structures of the complexes AlaRS−tRNA Ala / G3•U70 and AlaRS−tRNA Ala /A3•U70 suggest two very different conformations, representing a reactive and a nonreactive state, respectively. On the basis of these structures, it has been proposed that the G3•U70 base pair guides the −CCA end of the tRNA acceptor stem into the active site of AlaRS, thereby enabling aminoacylation. The crystal structures open up the possibility of directly computing the energetics of tRNA specificity by AlaRS. We have carried out molecular dynamics free-energy simulations to quantitatively estimate tRNA discrimination by AlaRS, focusing on the mutations of the single critical base pair G3•U70 to uncover the energetics underlying the accuracy of tRNA selection. The calculations show that the reactive complex is highly selective in favor of the cognate tRNA Ala /G3•U70 over its noncognate analogues (A3•U70/G3•C70/A3•C70). In contrast, the nonreactive complex is predicted to be unselective between tRNA Ala /G3•U70 and tRNA Ala /A3•U70. Utilizing our calculated relative binding free energies, we show how a simple three-step kinetic scheme for aminoacylation, involving both an initial nonspecific binding step and a subsequent transition to a selective reactive complex, accounts for the observed kinetics of the process.
Nucleic Acids Research, 1995
We describe a convenient, simple and novel continuous spectrophotometric method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate generated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphate, which in turn is used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methylpurine ribonucleoside. Of the reaction products, ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and hence provides a spectrophotometric signal that can be continuously followed. The nondestructive nature of the spectrophotometric assay allowed the re-use of the tRNAs in question in successive experiments. The usefulness of this method was demonstrated for glutaminyl-tRNA synthetase (GInRS) and tryptophanyl-tRNA synthetase. Initial velocities measured using this assay correlate closely with those assayed by quantitation of [3H]Gln-tRNA or [14C]Trp-tRNA formation respectively. In both cases amino acid transfer from the aminoacyl adenylate to the tRNA represents the rate determining step. In addition, aminoacyl adenylate formation by aspartyl-tRNA synthetase was followed and provided a more sensitive means of active site titration than existing techniques. Finally, this novel method was used to provide direct evidence for the cooperativity of tRNA and ATP binding to GInRS.
Journal of Molecular Biology, 2007
Tryptophanyl-tRNA synthetase (TrpRS) is a functionally dimeric ligase, which specifically couples hydrolysis of ATP to AMP and pyrophosphate to the formation of an ester bond between tryptophan and the cognate tRNA. TrpRS from Bacillus stearothermophilus binds the ATP analogue, adenosine-5′ tetraphosphate, AQP, competitively with ATP during pyrophosphate exchange. Estimates of binding affinity from this competitive inhibition and from isothermal titration calorimetry show that AQP binds 200 times more tightly than ATP both under conditions of inducedfit, where binding is coupled to an unfavourable conformational change, and under exchange conditions, where there is no conformational change. These binding data provide an indirect experimental measurement of +3.0 kcal/mole for the conformational free energy change associated with induced-fit assembly of the active site. Thermodynamic parameters derived from the calorimetry reveal very modest enthalpic changes, consistent with binding driven largely by a favorable entropy change. The 2.5 Å structure of the TrpRS:AQP complex, determined de novo by X-ray crystallography, resembles that of the previously described, pre-transition state TrpRS:ATP complexes. The anticodon-binding domain untwists relative to the Rossmann-fold domain by 20% of the way toward the orientation observed for the Products complex. An unexpected tetraphosphate conformation allows the γ̃ and δ̃ phosphate groups to occupy positions equivalent to those occupied by the β̃ and γ̃ phosphates of ATP. The β-phosphate effects a 1.11 Å extension that relocates the αphosphate toward the tryptophan carboxylate while the PPi mimic moves deeper into the KMSKS loop. This configuration improves interactions between enzyme and nucleotide significantly and uniformly in the adenosine and PPi binding subsites. A new hydrogen bond forms between S194 from the class I KMSKS signature sequence and the PPi mimic. These complementary thermodynamic and structural data are all consistent with the conclusion that the tetraphosphate mimics a transition-state in which the KMSKS loop develops increasingly tight bonds to the PPi leaving group, weakening linkage to the Pα as it is relocated by an energetically favourable domain movement. Consistent with extensive mutational data on Tyrosyl-tRNA synthetase, this aspect of ¶Corresponding author: Department of Biochemistry and Biophysics, CB 7260,
Proteins: Structure, Function, and Bioinformatics, 2010
A general approach to genetically encode unnatural amino acids (AA) with diverse chemical, biophysical, and biological properties into prokaryotes and eukaryotes was developed recently. 1-4 Before protein synthesis, each of the 20 standard amino acids (AA) must be attached to their specific tRNA molecule by a specific aminoacyl-tRNA synthetase (AA tRNA-RS). During protein synthesis, mRNA codons are recognized by a specific tRNA anticodon resulting in selective AA incorporation into the elongating protein chain in the ribosome. Aminoacyl-tRNA synthetase/tRNA pairs from archaea have been shown to be orthogonal to the endogenous AA tRNA-RS/AA tRNA pairs in E. coli, which means they do not interfere with any of the host pairs. In vivo incorporation of unnatural AA into proteins was facilitated in response to the amber codon TAG by AA tRNA-RS selectively charging the orthogonal tRNA with a specific unnatural AA. Using this approach, more than 30 unnatural AA have been cotranslationally incorporated into proteins with high fidelity and efficiency in vivo. 1-4 The identity of the AA is determined by the AA tRNA-RS specificity to covalently link a specific AA exclusively to its designated tRNA. This reaction involves several steps: selective binding of the AA and ATP to the synthetase, AA adenylation to activate the AA, selective binding of tRNA, and finally transfer of the adenylated AA to the 3 0 end of the tRNA forming the aminoacyl-tRNA via a covalent ester linkage between AA and tRNA. Experimentally, a directed evolution approach is used to alter the specificity of the orthogonal synthetase enzyme for the target unnatural AA. This is accomplished by randomizing the AA identity of 4-6 positions in the binding pocket. Libraries of enzyme variants comprising on the order of 10 9 mutants are passed through a series of positive and negative selection steps. Repeated rounds of positive and negative selection may result in the isolation of specific enzyme mutants that successfully incorporate target unnatural AA but not endogenous AA.
A Spectrophotometric Assay for Quantitative Measurement of Aminoacyl-tRNA Synthetase Activity
Journal of Biomolecular Screening, 2013
Aminoacyl-tRNA synthetases are enzymes that charge specific tRNAs with their cognate amino acids and play an essential role in the initial steps of protein synthesis. Because these enzymes are attractive targets for drug development in many microorganisms, there is a pressing need for assays suitable for compound screening. We developed (1) a high throughput assay for measuring aminoacyl-tRNA synthetase activity and (2) an accompanying method for preparing the tRNA substrate. The assay can be performed in 96-well plates and relies on malachite green detection of pyrophosphate (Pi) as an indicator of aminoacyl-tRNA synthetase activity. Analysis of Trypanosoma brucei isoleucyl-tRNA synthetase (IleRS) activity showed that the assay exhibits sensitivity to picomoles of product, and yielded a Z′ factor of 0.56. We show that this assay is applicable to other aminoacyl-tRNA synthetases and to enzyme inhibition studies. Using this assay, we found that the compound NSC616354 inhibits recombinant IleRS with an IC 50 of 0.6 µM. Enzymology studies were also performed with rIleRS and its K m and k cat determined as 3.97 x 10 -5 mol/L and 142 S -1 , respectively. This assay will facilitate the screening of compounds to identify inhibitors of aminoacyl-tRNA synthetases.
Analytical strategy for determination of active site sequences in aminoacyl-tRNA synthetases
Journal of Chromatography A, 1988
Affinity labelling with radioactive, periodate-oxidized tRNA has been used to investigate the structures of tRNA-binding sites in Escherichiu coli aminoacyl-tRNA synthetases. Labelled peptides were isolated by means of a combination of techniques involving chymotryptic digestion of the enzyme, gel filtration, ribonuclease digestion of tRNA, chromatography on a TSK 2000 column and reversed-phase chromatography. An isocratic phenylthiohydantoin identification system has been interfaced to a sequencer, allowing the characterization of modified lysine residues by means of both chromatographic retention and liquid scintillation counting.