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. 2008 May 9;283(19):12960-70.
doi: 10.1074/jbc.M801649200. Epub 2008 Mar 4.

Activation of D-tyrosine by Bacillus stearothermophilus tyrosyl-tRNA synthetase: 1. Pre-steady-state kinetic analysis reveals the mechanistic basis for the recognition of D-tyrosine

Anita Sheoran  1 , Gyanesh Sharma, Eric A First
Affiliations

Activation of D-tyrosine by Bacillus stearothermophilus tyrosyl-tRNA synthetase: 1. Pre-steady-state kinetic analysis reveals the mechanistic basis for the recognition of D-tyrosine

Anita Sheoran et al. J Biol Chem. .

Abstract

Tyrosyl-tRNA synthetase (TyrRS) is able to catalyze the transfer of both l- and d-tyrosine to the 3' end of tRNA(Tyr). Activation of either stereoisomer by ATP results in formation of an enzyme-bound tyrosyl-adenylate intermediate and is accompanied by a blue shift in the intrinsic fluorescence of the protein. Single turnover kinetics for the aminoacylation of tRNA(Tyr) by D-tyrosine were monitored using stopped-flow fluorescence spectroscopy. Bacillus stearothermophilus tyrosyl-tRNA synthetase binds d-tyrosine with an 8.5-fold lower affinity than that of l-tyrosine (K (D-Tyr)(d) = 102 microm) and exhibits a 3-fold decrease in the forward rate constant for the activation reaction (k (D-Tyr)(3) = 13 s(-1)). Furthermore, as is the case for l-tyrosine, tyrosyl-tRNA synthetase exhibits "half-of-the-sites" reactivity with respect to the binding and activation of D-tyrosine. Surprisingly, pyrophosphate binds to the TyrRS.d-Tyr-AMP intermediate with a 14-fold higher affinity than it binds to the TyrRS.l-Tyr-AMP intermediate (K (PPi)(d) = 0.043 for TyrRS.d-Tyr-AMP.PP(i)). tRNA(Tyr) binds with a slightly (2.3-fold) lower affinity to the TyrRS.d-Tyr-AMP intermediate than it does to the TyrRS.l-Tyr-AMP intermediate. The observation that the K (Tyr)(d) and k(3) values are similar for l- and d-tyrosine suggests that their side chains bind to tyrosyl-tRNA synthetase in similar orientations and that at least one of the carboxylate oxygen atoms in d-tyrosine is properly positioned for attack on the alpha-phosphate of ATP.

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Figures

FIGURE 1.
FIGURE 1.
Reaction diagram for the aminoacylation of tRNATyr by tyrosine. The activation of tyrosine by ATP and subsequent transfer of the tyrosyl moiety from the enzyme-bound tyrosyl-adenylate intermediate to the 3′ end of tRNATyr are shown. Dissociation and rate constants are shown above or below the step with which they are associated. Noncovalent interactions are indicated by “·” and covalent interactions are indicated by “-”.
FIGURE 2.
FIGURE 2.
Steady-state fluorescence emission spectra for B. stearothermophilus tyrosyl-tRNA synthetase in the presence of d-or l-tyrosine. The fluorescence emission spectra for B. stearothermophilus tyrosyl-tRNA synthetase in the absence and presence of d-tyrosine, l-tyrosine, MgATP, and disodium pyrophosphate are shown (λex = 295 nm, λem = 300–400 nm). The emission spectra of the enzyme (0.5 μm) in the presence of 200 μml-tyrosine and 10 mm MgATP (panels A and B), and 500 μmd-tyrosine and 10 mm MgATP (panels C and D), respectively, are shown. Panels A and C, tyrosine is added first followed by MgATP, and in panels B and D the respective orders of addition are reversed. With the exception of the TyrRS·ATP spectra (panels B and D), all of the emission spectra in which MgATP is present are corrected for the inner filter effects of MgATP by multiplying the spectra by a factor of 1.19. Panels E and F show changes in the relative fluorescence intensity (λex = 295 nm, λem = 300–400 nm) for the TyrRS·l-Tyr-AMP and TyrRS·d-Tyr-AMP complexes in the presence of 0.8 mm disodium pyrophosphate. The concentrations of TyrRS·l-Tyr-AMP and TyrRS·d-Tyr-AMP were 1 μm for these experiments. To ensure that formation of the enzyme-bound tyrosyl-adenylate complex is stoichiometric, all of the steady-state emission spectra were determined in the presence of inorganic pyrophosphatase.
FIGURE 3.
FIGURE 3.
Stopped-flow fluorescence emission spectra for the formation and pyrophosphorylation of the TyrRS·Tyr-AMP complex. Panel A shows the reaction trace for the formation of the TyrRS·d-Tyr-AMP complex, as determined by monitoring the decrease in the fluorescence emission above 320 nm. Tyrosyl-tRNA synthetase (0.5 μm) was preincubated in the presence of d-tyrosine and subsequently mixed with MgATP. Panel B shows the conversion of 0.25 μm TyrRS·d-Tyr-AMP + pyrophosphate to TyrRS + d-Tyr + ATP, determined by monitoring the increase in the fluorescence emission above 320 nm. Data acquisition for both curves was split, with 500 data points measured during the initial 20% of each reaction trace, and 500 data points measured during the remainder of the reaction trace.
FIGURE 4.
FIGURE 4.
Dissociation of tyrosine from the TyrRS·Tyr and TyrRS·Tyr·ATP complexes. The equilibrium constants for the dissociation of tyrosine from the TyrRS·Tyr (K Tyrd) and TyrRS·Tyr·ATP (K′ Tyrd) complexes were determined by fitting plots of kobs versus the concentration of d-tyrosine to Equation 4. For determination of K Tyrd, the ATP concentration was 0.5 mm, whereas for K′ Tyrd, it was 10 mm (K ATPd = 3.5 mm). Panels A and B show typical plots for kobs versus d-tyrosine concentration at 0.5 mm and 10 mm MgATP, respectively. The inset shows the data fit to the Eadie-Hofstee transformation of Equation 4.
FIGURE 5.
FIGURE 5.
Analysis of d-tyrosine binding by equilibrium dialysis. The binding of d-[14C]tyrosine to tyrosyl-tRNA synthetase during a typical equilibrium dialysis experiment is shown. Data are fit to the Langmuir isotherm (Equation 3) (24). The inset shows the data fit to the Scatchard equation (Equation 2) (23).
FIGURE 6.
FIGURE 6.
Analysis of MgATP binding to unliganded tyrosyl-tRNA synthetase. A typical plot for the dependence of kobs with respect to the concentration of MgATP in the presence of 10 μmd-tyrosine is shown. At this concentration of d-tyrosine, less than 10% of the enzyme contains d-tyrosine bound to the active site. The dissociation constant determined under these conditions corresponds to the dissociation of ATP from the TyrRS·ATP intermediate (i.e. K ATPd) (26).
FIGURE 7.
FIGURE 7.
Analysis of pyrophosphorolysis and pyrophosphate dissociation. A typical plot for the conversion of TyrRS·Tyr-AMP + pyrophosphate to TyrRS + Tyr + ATP is shown. The dissociation constant determined from this assay corresponds to the dissociation of pyrophosphate from the TyrRS·Tyr-AMP·PPi complex (K PPid), and the rate constant (k–3) corresponds to the rate constant for formation of the bond between the α- and β-phosphates of ATP.
FIGURE 8.
FIGURE 8.
Analysis of the tRNATyr aminoacylation reaction. A typical hyperbolic plot for the transfer of d-tyrosine from the TyrRS·Tyr-AMP intermediate to in vitro transcribed B. stearothermophilus tRNATyr substrate is shown. The forward rate constants (k4) and the tRNATyr dissociation (K tRNAd) constants were determined from a plot of initial rate versus tRNATyr concentration.
FIGURE 9.
FIGURE 9.
Standard free energy diagram for the aminoacylation of tRNATyr by l- and d-tyrosine for B. stearothermophilus tyrosyl-tRNA synthetase. Solid and dashed lines indicate the standard free energy changes during the course of the reaction for the activation of d-tyrosine and l-tyrosine, respectively, and their subsequent transfer to tRNATyr. Standard free energy values for the enzyme with l-tyrosine are taken from Refs. and . For the activation of l-tyrosine, K TyrdK′ ATPd was used in place of the K ATPdK′ Tyrd term in Equations 7, 8, 9, 10.
FIGURE 10.
FIGURE 10.
Modeling the TyrRS·d-Tyr and TrpRS·d-Trp complexes. Panel A, docking of l- and d-tyrosine to B. stearothermophilus tyrosyl-tRNA synthetase is shown. l- and d-tyrosine are shown as stick models with oxygen atoms in red, nitrogen atoms in blue, and carbon atoms in green (l-tyrosine) and cyan (d-tyrosine). B. stearothermophilus tyrosyl-tRNA synthetase is shown as a schematic representation (magenta). Docking was done using Autodock 4.0 as described in the text. Panel B, docking of l- and d-tryptophan to B. stearothermophilus tryptophanyl-tRNA synthetase is shown. l- and d-tryptophan are shown as stick models with oxygen atoms in red, nitrogen atoms in blue, and carbon atoms in green (l-tryptophan) and cyan (d-tryptophan). B. stearothermophilus tryptophanyl-tRNA synthetase is shown as a schematic representation with the Gln-147 side chain shown as a stick model (magenta). Modeling was done by superimposing the d-tryptophan coordinates onto those of l-tryptophan and selecting the rotomer that most closely resembled the conformation adopted by d-tryptophan in the TrpRS·d-Trp complex as described in the text. The molecular graphics for this figure were generated using PyMol (44).
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