Ammonium Scanning in an Enzyme Active Site

Thompson, Damien; Lazennec, Christine; Plateau, Pierre; Simonson, Thomas

doi:10.1074/jbc.m704788200

Cited by 24 publications

(16 citation statements)

References 60 publications

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“…This also shows that the absolute configuration of the amino acid is not required for recognition inside the active site of AspRS, and most probably the same holds for other aaRSs. This finding matches with the results of Thompson et al [24] who concluded that there is only limited chiral specificity for L-Asp, leading to an esterification of (D)-Asp to tRNA Asp with a rate of 1:4000 for (D)-Asp vs (L)-Asp.…”

Section: Discussionsupporting

confidence: 92%

“…From their [24]theoretical studies they concluded that the network of electrostatic interactions between the incoming amino acid, ATP and the synthetase are highly unfavorable for incorporation of a (D)-amino acid. Not only in the aminoacylation step (relevant for our studies), but likewise in the peptide bond formation reactions, it would be virtually impossible to incorporate (D)-amino acids in protein structures.…”

Section: Discussionmentioning

confidence: 99%

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N-Alkylated Aminoacyl sulfamoyladenosines as Potential Inhibitors of Aminoacylation Reactions and Microcin C Analogues Containing D-Amino Acids

et al. 2013

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Microcin C analogues were recently envisaged as important compounds for the development of novel antibiotics. Two issues that may pose problems to these potential antibiotics are possible acquisition of resistance through acetylation and in vivo instability of the peptide chain. N-methylated aminoacyl sulfamoyladenosines were synthesized to investigate their potential as aminoacyl tRNA synthetase inhibitors and to establish whether these N-alkylated analogues would escape the natural inactivation mechanism via acetylation of the alpha amine. It was shown however, that these compounds are not able to effectively inhibit their respective aminoacyl tRNA synthetase. In addition, we showed that (D)-aspartyl-sulfamoyladenosine (i.e. with a (D)-configuration for the aspartyl moiety), is a potent inhibitor of aspartyl tRNA synthetase. However, we also showed that the inhibitory effect of (D)- aspartyl-sulfamoyladenosine is relatively short-lasting. Microcin C analogues with (D)-amino acids throughout from positions two to six proved inactive. They were shown to be resistant against metabolism by the different peptidases and therefore not able to release the active moiety. This observation could not be reversed by incorporation of (L)-amino acids at position six, showing that none of the available peptidases exhibit endopeptidase activity.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

N-Alkylated Aminoacyl sulfamoyladenosines as Potential Inhibitors of Aminoacylation Reactions and Microcin C Analogues Containing D-Amino Acids

et al. 2013

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“…Recently published molecular dynamics simulations predict that the free energy difference between the binding of L-and D-tyrosine to E. coli tyrosyl-tRNA synthetase is 13 (Ϯ8) kJ/mol (41). Both our results and those of Thompson et al (41) are close to the experimentally determined free energy difference for the binding of L-and D-tyrosine (⌬⌬G 0 ϭ 5.0 kJ/mol).…”

Section: Discussionsupporting

confidence: 78%

Activation of d-Tyrosine by Bacillus stearothermophilus Tyrosyl-tRNA Synthetase

Sheoran

Sharma

First

2008

Journal of Biological Chemistry

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Tyrosyl-tRNA synthetase (TyrRS) 2 catalyzes the transfer of tyrosine to the 3Ј end of tRNA Tyr in a two-step reaction (Fig. 1). In the first step, tyrosine is activated by ATP, forming the enzyme-bound tyrosyl-adenylate intermediate. In the second step, the tyrosyl moiety is transferred to the 3Ј end of tRNA Tyr . The observations that the two steps of the reaction can be run independently of each other, and that formation of the tyrosyladenylate intermediate is accompanied by a change in the intrinsic fluorescence of the enzyme, make it possible to use stopped-flow fluorescence to monitor single turnover kinetics for each step in the reaction (2, 3).Tyrosyl-tRNA synthetase is composed of two identical 47-kDa subunits, each of which consists of a Rossmann fold domain containing the active site, a helical anticodon binding domain, and a carboxyl-terminal domain that binds the variable loop in tRNA Tyr . Tyrosyl-tRNA synthetase exhibits an extreme form of negative cooperativity with respect to tyrosine binding, known as "half-of-the-sites" reactivity, in which the unliganded subunit is completely inactivated. This behavior has been rationalized by the observation that, in solution, tyrosyltRNA synthetase binds only one molecule of tRNA Tyr and therefore has no need for two functional active sites. Discrimination between L-tyrosine and other amino acids is achieved solely on the basis of binding affinity (i.e. there is no editing domain in tyrosyl-tRNA synthetase). Surprisingly, Calendar and Berg (4) observed that tyrosyl-tRNA synthetase is able to aminoacylate tRNA with either the L-or D-stereoisomer of tyrosine, although activation is more efficient for L-tyrosine than it is for D-tyrosine. Hydrolysis of D-Tyr-tRNA Tyr is catalyzed by D-tyrosyl-tRNA deacylase in vivo, as tyrosyl-tRNA synthetase does not have an editing mechanism to prevent formation of D-Tyr-tRNA Tyr (5-7).Recognition of tRNA Tyr differs between bacteria and eukaryotes, with the bacterial and eukaryotic (or archaeal) tyrosyl-tRNA synthetases being unable to efficiently aminoacylate each others' tRNA Tyr substrates (8,9). This property has been exploited to introduce unnatural amino acids into proteins in both bacterial and eukaryotic systems. For example, Schultz and co-workers (10) have modified the tyrosyl-tRNA synthetase:tRNA Tyr pair from Methanococcus jannaschii so that it is completely nonorthologous to that of Escherichia coli. By replacing the anticodon in tRNA Tyr with one that is complementary to a stop codon, they have been able to introduce unnatural amino acids at specific positions in recombinant proteins expressed from E. coli (10). The observation that tyrosyltRNA synthetase catalyzes the aminoacylation of tRNA Tyr by D-tyrosine raises the possibility that tyrosyl-tRNA synthetase variants designed to incorporate unnatural L-amino acids into proteins can be adapted to selectively incorporate the D-analogs of the unnatural amino acids. As a first step toward this goal, we have characterized the binding, activation, and transfer of D...

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“…6 7 The residues composing the nanospace of the active site stabilize the substrates and favor the progress of the reaction by catalysis. 8 Extensive studies about the active site of aaRSs are carried out, principally using crystallographic methods complemented by biochemical methods, spectroscopic studies, computer simulations [33][34][35][36][37][38][39][40] and electronic structure based analysis. [41][42][43][44][45][46] Despite the wealth of information, the understanding of the principles controlling the organization of the active sites is yet to be completed.…”

Section: Introductionmentioning

confidence: 99%

Active Site Nanospace of Aminoacyl tRNA Synthetase: Difference Between the Class I and Class II Synthetases

Dutta¹,

Choudhury²,

Banik³

et al. 2014

j nanosci nanotechnol

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The present work is aimed at understanding the origin of the difference in the molecular organization of the active site nanospaces of the class I and class II aminoacyl tRNA synthetases (aaRSs) which are tunnel-like structures. The active site encloses the cognate amino acid (AA) and the adenosine triphosphate (ATP) to carry out aminoacylation reaction. Comparison of the structures of the active site of the class I and class II (aaRSs) shows that the nanodimensional tunnels are curved in opposite directions in the two classes. We investigated the origin of this difference using quantum mechanical computation of electrostatic potential (ESP) of substrates, surrounding residues and ions, using Atoms in Molecule (AIM) Theory and charge population analysis. We show that the difference is principally due to the variation in the spatial charge distribution of ATP in the two classes which correspond to extended and bent conformations of ATP. The present computation shows that the most feasible pathway for nucleophilic attack to alphaP is oppositely directed for class I and class II aaRSs. The available crystal structures show that the cognate AA is indeed located along the channel favorable for nucleophilic attack as predicted by the ESP analysis. It is also shown that the direction of the channel changes its orientation when the orientation of ATP is changed from extended to a bent like structure. We further used the AIM theory to confirm the direction of the approach of AA in each case and the results corroborate the results from the ESP analysis. The opposite curvatures of the active site nanospaces in class I and class II aaRSs are related with the influence of the charge distributions of the extended and bent conformations of ATP, respectively. The results of the computation of electrostatic potential by successive addition of active site residues show that their roles on the reaction are similar in both classes despite the difference in the organization of the active sites of class I and class II aaRSs. The difference in mechanism in two classes as pointed out in recent study (S. Dutta Banik and N. Nandi, J. Biomol. Struct. Dyn. 30, 701 (2012)) is related with the fact that the relative arrangement of the ATP with respect to the AA is opposite in class I and class II aaRSs as explained in the present work. The charge population difference between the reacting centers (which are the alphaP atom of ATP (q(p)) and the attacking oxygen atom of carboxylic acid group (q(o)), respectively) denoted by delta(q), is a measure of the propensity of nucleophilic attack. The population analysis of the substrate AA shows that a non-negligible difference exists between the attacking oxygens of AA in class I (syn) and in class II (anti) which is one reason for the lower value of delta(q) in class II relative to class I. The population analysis of the AA, ATP, Mg+2 ions and active site residues shows that the difference in delta(q) values of the two classes is substantially reduced. When ions and residues are considered. Thus, the...

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Ammonium Scanning in an Enzyme Active Site

Cited by 24 publications

References 60 publications

N-Alkylated Aminoacyl sulfamoyladenosines as Potential Inhibitors of Aminoacylation Reactions and Microcin C Analogues Containing D-Amino Acids

N-Alkylated Aminoacyl sulfamoyladenosines as Potential Inhibitors of Aminoacylation Reactions and Microcin C Analogues Containing D-Amino Acids

Activation of d-Tyrosine by Bacillus stearothermophilus Tyrosyl-tRNA Synthetase

Active Site Nanospace of Aminoacyl tRNA Synthetase: Difference Between the Class I and Class II Synthetases

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