Lysyl-tRNA Synthetase from Bacillus stearothermophilus: The Trp314 Residue is Shielded in a Non-polar Environment and is Responsible for the Fluorescence Changes Observed in the Amino Acid Activation Reaction

Takita, Teisuke; Nakagoshi, Makoto; Inouye, Kuniyo; Tonomura, Ben’ichiro

doi:10.1016/s0022-2836(02)01238-x

Cited by 9 publications

(3 citation statements)

References 53 publications

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“…The good agreement supports both the simulation model and the close relation between the K m and K d ratios for this enzyme. For the homologous class IIb LysRS, the Lys dissociation constant is known to be well approximated by the Michaelis constant (64). The TyrRS control simulations are also consistent with the experiment.…”

Section: Discussionsupporting

confidence: 68%

“…Furthermore, if we make the rough assumption that the chemical turnover steps are similar for L-Asp and D-Asp, then the different rates for L-Asp and D-Asp are mainly governed by the ratio of their Michaelis constants. Although K m is not identical to a dissociation constant in general, for the homologous class IIb LysRS, the lysine dissociation constant (27 M) can be accurately approximated by its Michaelis constant (9 M for tRNA aminoacylation and 16 M for ATP/PPi exchange) (64). For AspRS, similarly, the ratio of the L-Asp and D-Asp acylation rates should approximate at least roughly the ratio of their dissociation constants.…”

Section: L-asp Versus D-asp Bindingmentioning

confidence: 99%

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Ammonium Scanning in an Enzyme Active Site

Thompson

Lazennec

Plateau

et al. 2007

Journal of Biological Chemistry

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The aminoacyl-tRNA synthetases (aaRSs) 3 play a crucial role in preserving the accuracy of genetic code translation, linking each amino acid to a cognate tRNA, which carries the corresponding anticodon. Each of the 20 aaRSs must bind its target amino acid substrate with a high specificity (1-8). In the "class IIb" subgroup, formed of AspRS (9 -16, 21-26), AsnRS (17,24),25), a network of electrostatic interactions ensures binding specificity for the cognate amino acid side chain in each case. Experimental and theoretical studies show, for example, that the preference of AspRS for Asp over its neutral analogue, Asn, involves a complex mechanism (24, 25). Proton binding, ATP binding, and long-range electrostatic interactions play important roles, and net positive electrostatic potential in the active site permits AspRS to bind L-Asp much more strongly than L-Asn.The translation apparatus must also preserve the homochirality of proteins, and aaRSs should be specific for L-amino acids. Kinetic experiments show, however, that AspRS does not strongly distinguish between the "left-handed" L-Asp and "right-handed" D-Asp stereoisomers. Indeed, D-Asp-tRNA Asp is produced at detectable levels by Escherichia coli AspRS (26), at a rate only 4,000 times lower than L-Asp-tRNA Asp . To preserve homochirality in vivo (27), editing (28) of D-Asp-tRNA Asp by a D-aminoacyl-tRNA deacylase is performed (26). For biotechnology applications, it is not always desirable to preserve homochirality, and D-amino acids are of great potential interest in protein engineering and design. For example, mixed L-and D-amino acid proteins are used in the development of synthetic vaccines (29,30). It is hence of interest, both from a fundamental and an engineering perspective, to better understand how aaRSs distinguish between L-and D-amino acids.In the present work, we use simulations and experiments to investigate the origins of chiral specificity of E. coli AspRS for its L-aspartate substrate (L-Asp). Asp has a pseudosymmetry, broken only by its ammonium group, and so the enzyme must protect not only against D-Asp, but against an alternate, "inverted" orientation where the two substrate carboxylates are swapped (Fig. 1A). The inverted orientation is seen in the crystal structure of a homologous enzyme, asparagine synthetase (31). We compare both L-Asp and D-Asp, in regular and inverted orientations, and the metabolite succinate, where the ammonium group is removed altogether and the ligand has an additional negative charge. This is done with a state of the art molecular dynamics free energy (MDFE) technique (21)(22)(23)(24)(25)(32)(33)(34)(35)(36)(37)(38)(39), by computing the change in binding free energy when an ammonium group is inserted at either possible position on either succinate methylene group.By scanning all possible ammonium positions on the ligand, we can also address an interesting, secondary question: the strength of electrostatic interactions in the active site. Simulations have played a significant role in establishing the impor-

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Section: Discussionsupporting

confidence: 68%

Section: L-asp Versus D-asp Bindingmentioning

confidence: 99%

Ammonium Scanning in an Enzyme Active Site

Thompson

Lazennec

Plateau

et al. 2007

Journal of Biological Chemistry

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“…A survey of those systems where both detailed kinetics and structural analysis have been conducted reinforces this point. Among the class II systems, considerable structural evidence suggests the importance of induced fit for the histidyl (13,14,55), prolyl (14), and lysyl (11,56,57) enzymes which, as described above for ThrRS, exhibit analogous amino acid-induced conformational changes. Notably, the pre-steady state kinetic of LysRS was also interpreted as a two-step mechanism in which rapid equilibrium binding of lysine and ATP was followed by a unimolecular conformational change (43).…”

Section: Development Of a Kinetic Schemementioning

confidence: 89%

Induced Fit and Kinetic Mechanism of Adenylation Catalyzed by Escherichia coli Threonyl-tRNA Synthetase

2003

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Threonyl-tRNA synthetase (ThrRS) must discriminate among closely related amino acids to maintain the fidelity of protein synthesis. Here, a pre-steady state kinetic analysis of the ThRS-catalyzed adenylation reaction was carried out by monitoring changes in intrinsic tryptophan fluorescence. Stopped flow fluorimetry for the forward reaction gave a saturable fluorescence quench whose apparent rate increased hyperbolically with ATP concentration, consistent with a two-step mechanism in which rapid substrate binding precedes an isomerization step. From similar experiments, the equilibrium dissociation constants for dissociation of ATP from the E.Thr complex (K(3) = 450 +/- 180 microM) and threonine from the E.ATP complex (K'(4) = 135 microM) and the forward rate constant for adenylation (k(+5) = 29 +/- 4 s(-1)) were determined. A saturable fluorescence increase accompanied the pyrophosphorolysis of the E.Thr - AMP complex, affording the dissociation constant for PP(i) (K(6) = 170 +/- 50 microM) and the reverse rate constant (k(-5) = 47 +/- 4 s(-1)). The longer side chain of beta-hydroxynorvaline increased the apparent dissociation constant (K(4[HNV]) = 6.8 +/- 2.8 mM) with only a small reduction in the forward rate (k'(+5[HNV]) = 20 +/- 3.1 s(-1)). In contrast, two nonproductive substrates, threoninol and the adenylate analogue 5'-O-[N-(L-threonyl)sulfamoyl]adenosine (Thr-AMS), exhibited linear increases in k(app) with ligand concentration, suggesting that their binding is slow relative to isomerization. The proposed mechanism is consistent with steady state kinetic parameters. The role of threonine binding loop residue Trp434 in fluorescence changes was established by mutagenesis. The combined kinetic and molecular genetic analyses presented here support the principle of induced fit in the ThrRS-catalyzed adenylation reaction, in which substrate binding drives conformational changes that orient substrates and active site groups for catalysis.

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Protein Fluorescence

2006

Principles of Fluorescence Spectroscopy

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Lysyl-tRNA Synthetase from Bacillus stearothermophilus: The Trp314 Residue is Shielded in a Non-polar Environment and is Responsible for the Fluorescence Changes Observed in the Amino Acid Activation Reaction

Cited by 9 publications

References 53 publications

Ammonium Scanning in an Enzyme Active Site

Ammonium Scanning in an Enzyme Active Site

Induced Fit and Kinetic Mechanism of Adenylation Catalyzed by Escherichia coli Threonyl-tRNA Synthetase

Protein Fluorescence

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