Altering the Enantioselectivity of Tyrosyl-tRNA Synthetase by Insertion

Feb 18, 2016 - Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center in Shreveport, 1501 Kings Highway, ...
0 downloads 0 Views 504KB Size
Download PDF
Article pubs.acs.org/biochemistry

Altering the Enantioselectivity of Tyrosyl-tRNA Synthetase by Insertion of a Stereospecific Editing Domain Charles J. Richardson∥ and Eric A. First* Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center in Shreveport, 1501 Kings Highway, Shreveport, Louisiana 71130, United States S Supporting Information *

ABSTRACT: Translation of mRNAs by the ribosome is stereospecific, with only L-amino acids being incorporated into the nascent polypeptide chain. This stereospecificity results from the exclusion of D-amino acids at three steps during protein synthesis: (1) the aminoacylation of tRNA by aminoacyl-tRNA synthetases, (2) binding of aminoacyl-tRNAs to EF-Tu, and (3) recognition of aminoacyl-tRNAs by the ribosome. As a first step toward incorporating D-amino acids during protein synthesis, we have altered the enantioselectivity of tyrosyl-tRNA synthetase. This enzyme is unusual among aminoacyl-tRNA synthetases, as it can aminoacylate tRNA with D-tyrosine (albeit at a reduced rate compared to L-tyrosine). To change the enantioselectivity of tyrosyl-tRNA synthetase, we introduced the post-transfer editing domain from Pyrococcus horikoshii phenylalanyl-tRNA synthetase into the connective polypeptide 1 (CP1) domain of Geobacillus stearothermophilus tyrosyl-tRNA synthetase (henceforth designated TyrRS-FRSed). We show that the phenylalanyl-tRNA synthetase editing domain is stereospecific, hydrolyzing L-Tyr-tRNATyr, but not D-Tyr-tRNATyr. We further show that inserting the phenylalanyl-tRNA synthetase editing domain into the CP1 domain of tyrosyl-tRNA synthetase decreases the activity of the synthetic site in tyrosyltRNA synthetase. This decrease in activity is critical, as it prevents the rate of synthesis from overwhelming the ability of the editing domain to hydrolyze the L-Tyr-tRNATyr product. Overall, inserting the phenylalanyl-tRNA synthetase editing domain results in a 2-fold shift in the enantioselectivity of tyrosyl-tRNA synthetase toward the D-Tyr-tRNATyr product. When a 4-fold excess of D-tyrosine is used, approximately 40% of the tRNATyr is aminoacylated with D-tyrosine.

E

acids. As a result, D-peptides fail to stimulate either the proliferation of helper T cells or the cytotoxic effects of killer T cells, decreasing their immunogenicity.3 Lastly, as tissues age, they display an increase in proteins containing D-amino acids, including the eye lens crystallins, myelin basic proteins, erythrocyte proteins, and β-amyloid peptides from Alzheimer’s disease brain tissue.4−6 The ability to readily synthesize these proteins would aid in efforts to understand their roles in disease. Ideally, one would like to combine the diversity available in nonribosomal peptide synthesis with the efficiency and flexibility of ribosomal protein synthesis to generate novel proteins with unique catalytic functions, physical properties, and chemical characteristics. This has led to the development of methods that expand the genetic code to allow the incorporation of unnatural amino acids into proteins.7,8 Unfortunately, there are several roadblocks to incorporating D-amino acids into proteins with these methods. First, the aminoacyl-tRNA synthetaseswhich catalyze the attachment of amino acids to their cognate tRNAsare stereospecific, with most aminoacyl-tRNA synthetases unable to use D-amino acids.9,10 Although this limitation can be partially overcome through the use of artificially evolved aminoacyl-tRNA

nzymes catalyze chemical reactions with exquisite specificity and enantioselectivity. While RNA does act as a catalyst in nature, the greater chemical diversity found among the 20 naturally occurring amino acids has allowed proteins to dominate as catalysts in all known organisms. Although this chemical diversity has generated a vast number of conformations and active site configurations in proteins, ribosomal protein synthesis is generally limited to the production of homochiral polypeptides composed of 19 L-amino acids and glycine. In nature, this limitation is overcome through the use of either nonribosomal peptide synthesis or post-translational modification, allowing a wider diversity of amino acids, including D-amino acids, to be incorporated into peptides. While useful in nature, from a synthetic biology perspective these approaches are less than ideal as introducing D-amino acids at multiple positions would require distinct enzymes for each position in the peptide. The ability to incorporate D-amino acids into peptides and proteins has a number of advantages. Peptides (and proteins) with D-amino acids are able to adopt unique structures that are either unavailable to peptides composed solely of L-amino acids or are less stable than peptides containing one or more D-amino acids.1,2 In addition, since they are not efficiently recognized and cleaved by proteases, peptides containing D-amino acids tend to resist proteolysis, increasing their biological half-lives. Furthermore, the inability of proteases to recognize the Dstereoisomer prevents the major histocompatibility complex from processing and presenting peptides containing D-amino © 2016 American Chemical Society

Received: October 26, 2015 Revised: January 24, 2016 Published: February 18, 2016 1541

DOI: 10.1021/acs.biochem.5b01167 Biochemistry 2016, 55, 1541−1553

Article

Biochemistry

compete with L-tyrosine is increased 2-fold compared to that of the wild-type enzyme when equimolar concentrations of L- and D-tyrosine are present. When a 4-fold excess of D-tyrosine is used, approximately 40% of the tRNATyr is aminoacylated with D-tyrosine.

synthetase-like ribozymes (flexizymes), this technology requires the synthesis of activated amino acids and preacylation of the tRNA substrate, restricting its use to in vitro peptide synthesis.11,12 Second, EF-Tuwhich transports aminoacyltRNAs to the ribosomeis unable to bind D-aminoacyltRNAs.13−16 Third, although the ribosome can use D-aminoacyl-tRNAs in translating the mRNA sequence, the efficiency is much lower than that of L-aminoacyl-tRNAs.17−19 This last limitation has been partially alleviated by mutations in the peptidyl-transferase center and 23S rRNA helix 89 of the ribosome.20,21 Furthermore, recent analysis into the mechanism by which the translational machinery discriminates against Daminoacyl-tRNAs indicates that mutations in the ribosome exit tunnel entrance may further enhance the ribosome’s ability to incorporate D-amino acids into proteins.22 These observations suggest that the stereospecificity of the aminoacyl-tRNA synthetases and EF-Tu are the limiting factors in engineering the translational machinery to use D-amino acids. Eliminating one of these limiting factors (e.g., the stereospecificity of aminoacyl-tRNA synthetases) would allow a genetic selection approach to be used to overcome the other roadblock (e.g., the stereospecificity of EF-Tu). As a first step toward engineering the translational machinery to use D-amino acids, we have begun efforts to alter the enantioselectivity of tyrosyl-tRNA synthetase. Tyrosyl-tRNA synthetase is a homodimer composed of four domains: an amino-terminal Rossmann fold domain containing the active site, the connective polypeptide 1 (CP1) domain which is inserted between the two halves of the Rossmann fold domain and forms the dimer interface, an α-helical “hinge” domain which interacts with the anticodon in tRNATyr, and a carboxylterminal domain which interacts with the anticodon stem and variable loop in tRNA.23−26 Tyrosyl-tRNA synthetase does not contain an editing domain, as active site selectivity is sufficiently high to discriminate between L-tyrosine and noncognate amino acids. Furthermore, tyrosyl-tRNA synthetase is one of five aminoacyl-tRNA synthetases that has been found to aminoacylate tRNA with both L- and D-amino acids.10 In the case of tyrosyl-tRNA synthetase, the efficiency of tRNA aminoacylation by D-tyrosine is reduced 30-fold relative to that of Ltyrosine.27−29 In this paper, we test the hypothesis that introducing a stereospecific editing domain is sufficient to alter the enantioselectivity of tyrosyl-tRNA synthetase. Specifically, we introduced the editing domain from Pyrococcus horikoshii phenylalanyl-tRNA synthetase, which hydrolyzes tyrosyltRNA, into the CP1 domain of Geobacillus stearothermophilus tyrosyl-tRNA synthetase (henceforth referred to as the TyrRSFRSed variant). The choice of the P. horikoshii editing domain, as well as its insertion into the CP1 domain of tyrosyl-tRNA synthetase, is based on previous investigations.30 Specifically, Yokoyama and colleagues demonstrated that insertion of the P. horikoshii editing domain into iodo-tyrosyl-tRNA synthetase (a tyrosyl-tRNA synthetase variant that aminoacylates tRNA with 3-iodo-L-tyrosine) selectively hydrolyzes L-tyrosyl-tRNA, but not 3-iodo-L-tyrosyl-tRNA. In this paper, we show that insertion of the editing domain into tyrosyl-tRNA synthetase results in (1) the stereospecific hydrolysis of L-tyrosyl-tRNATyr, but not D-tyrosyl-tRNATyr, (2) a decrease in the binding affinity of tyrosyl-tRNA synthetase for the tyrosyl-adenylate intermediate, and (3) a decrease in the rate of formation for the tyrosyl-tRNA Tyr product. Taken together, these effects produced an enzyme in which the ability of D-tyrosine to

■

MATERIALS AND METHODS Materials. Materials were obtained from the following sources: TOPO TA cloning kit (Life Technologies), pGEM-T Easy cloning kit (Promega), pET30a(+) expression vector and Escherichia coli BL21 (DE3) and Rosetta 2 (DE3) cells (EMD Biosciences), E. coli XL2 Blue cells (Agilent Technologies), plasmid miniprep purification kit (Omega Bio-Tek), T4 DNA ligase and FokI, NdeI, NheI, BspEI, and XhoI endonucleases (New England Biolabs), Taq DNA polymerase (G-Biosciences), Biosafe II scintillation cocktail (Research Products International Corporation, Mount Prospect, IL), Dispo Equilibrium Dialyzers (Harvard Apparatus, Holliston, MA), [14C]-L-tyrosine (Moravek, Brea, CA), and [14C] and [3H]-Dtyrosine (American Radiochemicals, St. Louis, MO). DNA synthesis was performed by Genscript Inc. (Piscataway, NJ). All other reagents were obtained from either VWR International (Radnor, PA) or Fisher Scientific (Pittsburgh, PA). DNA sequencing was performed by the DNA lab at Arizona State University (Tempe, AZ). Curve fitting and graphing were performed using GraFit v 5.0.6 (Erithacus Software, Ltd., London, UK) and Kaleidograph v 3.6 (Synergy Software, Reading, PA). Subcloning the Phenylalanyl-tRNA Synthetase Editing Domain into the CP1 Domain of G. stearothermophilus Tyrosyl-tRNA Synthetase. Construction of a plasmid for the expression of wild-type G. stearothermophilus tyrosyl-tRNA synthetase, pYTS5-WT, has previously been described.31 To insert the phenylalanyl-tRNA synthetase editing domain into tyrosyl-tRNA synthetase, BspEI, XbaI, and NheI restriction sites were introduced between residues corresponding to glycine 161 and isoleucine 162 in the connective peptide 1 (CP1) domain of the tyrosyl-tRNA synthetase coding sequence using overlap extension PCR. This plasmid is designated pGEM-YTS-CP1ins. The tyrosyl-tRNA synthetase coding sequence was then removed from pGEM-YTS-CP1ins by digestion with BamHI and SalI and inserted into the pYTS5WT vector in place of the wild-type tyrosyl-tRNA synthetase coding sequence. This plasmid is designated pYTS7-CP1ins. The P. horikoshii phenylalanyl-tRNA synthetase editing domain coding sequence (residues 83 through 275 from the phenylalanyl-tRNA synthetase β-subunit) was synthesized by Genscript Inc., such that it contains an eight-codon linker (Gly-SerAla-Ser-Pro-Asp-Ser-Ala) at the 3′ end, and BspE1 and NheI restriction sites at the 5′ and 3′ ends, respectively. This sequence was subcloned into the pYTS7-CP1ins plasmid via the BspEI and NheI restriction sites. The resulting tyrosyltRNA synthetase-phenylalanyl-tRNA synthetase editing domain chimera (TyrRS-FRSed) was PCR-amplified and subcloned into the pET30a(+) expression vector, such that the TyrRSFRSed coding sequence is in frame with a sequence encoding a carboxyl-terminal 6xHis-tag. This clone is designated pYF1WT. Substitution of asparagine 217 by alanine (N217A) in the phenylalanyl-tRNA synthetase editing domain was achieved by two-step overlapping PCR of the TyrRS-FRSed coding sequence in pYF1-WT.32 This clone is designated pYF1N217A. 1542

DOI: 10.1021/acs.biochem.5b01167 Biochemistry 2016, 55, 1541−1553

Article

Biochemistry

fluorescence on formation of the transition state for tyrosine activation.29,45,46 Single-turnover kinetics for the TyrRS-WT and TyrRS-FRSed variants were monitored by following the decrease in fluorescence above 320 nm (λex = 295 nm) using an Applied Photophysics SX-18.MV spectrophotometer as previously described.45,47 All assays were conducted in 144 mM Tris, 10 mM MgCl2, pH 7.78 at 25 °C. The observed rate constant for each substrate concentration was determined by fitting the fluorescence traces to a single exponential with floating end point equation:45

Protein Expression and Purification. Saccharomyces cerevisiae AMP deaminase, S. cerevisiae IMP dehydrogenase, S. cerevisiae inorganic pyrophosphatase, S. cerevisiae phenylalanyltRNA synthetase, Mycobacterium tuberculosis cyclodityrosine synthase, Thermus thermophilus D-tyrosyl-tRNA deacylase, G. stearothermophilus tyrosyl-tRNA synthetase, and T7 RNA polymerase were expressed as recombinant proteins in either BL21 DE3 or Rosetta 2 DE3 E. coli cells and purified by NiNTA affinity chromatography as previously described.33−36 Expression and purification of the recombinant TyrRS-FRSed chimera were performed using the protocol described for purification of the wild type tyrosyl-tRNA synthetase.35 The TyrRS-FRSed protein was purified to >95% homogeneity based on SDS-PAGE, and its concentration was determined based on its absorbance at 280 nm (ε280 = 145 540 M−1 cm−1 for the TyrRS-FRSed homodimer, as calculated using the ExPASy ProtParam tool).37 Purified proteins were stored in 20 mM Tris, 10 mM β-mercaptoethanol, 1 mM EDTA, 20% glycerol (v/v), pH 7.8 at −70 °C. The TyrRS-FRSed-N217A variant was isolated and stored using the same procedure. In Vitro Transcription and Purification of tRNATyr. In vivo, G. stearothermophilus tRNATyr contains modified nucleotides (e.g., queuosine and 2-(methylthio)-N6-isopentenyladenosine at positions 34 and 37); however, these modifications do not affect the aminoacylation of tRNATyr by tyrosyl-tRNA synthetase.38,39 As unmodified tRNATyr is kinetically equivalent to modified tRNATyr, G. stearothermophilus tRNATyr was synthesized by runoff in vitro transcription of FokI digested pGFX-WT using T7 RNA polymerase, as previously described.40 After purification, tRNA was resuspended in buffer containing 100 mM HEPES, pH 7.5 at a final concentration of 15−20 μM (based on A260 measurements, ε260 = 806 100 M−1 cm−1) and annealed by heating the tRNA at 85 °C for 5 min, adding MgCl2 to a final concentration of 10 mM, and slowly cooling to room temperature in a heat block (>2 h).41 The aminoacylation of tRNATyr was quantified using a nitrocellulose filter assay in which the incorporation of [14C]-Ltyrosine into the L-Tyr-tRNATyr product is monitored.38 Comparing the incorporation of [14C]-L-tyrosine into the LTyr-tRNATyr with the concentration of tRNATyr determined from the A260 measurements indicated that >70% of the tRNATyr was aminoacylated.42,43 Active Site Titration of Tyrosyl-tRNA Synthetase. The fraction of wild-type tyrosyl-tRNA synthetase (TyrRS-WT) and TyrRS-FRSed that is active was determined using an active site titration assay that monitors the incorporation of [14C]-Ltyrosine into the enzyme-bound tyrosyl-adenylate intermediate (TyrRS·Tyr-AMP).44 In this assay, TyrRS-WT or TyrRSFRSed (1−5 μM) is incubated with 50 mM Tris, pH 7.8, 10 mM MgCl2, 10 mM MgATP, 10 μM [14C]-L-tyrosine, and 1 U/ mL inorganic pyrophosphate. At 2′, 5′, and 10′ time points, aliquots are removed and filtered through nitrocellulose discs to separate the TyrRS·[14C]-L-Tyr-AMP intermediate from unbound [14C]-L-tyrosine. The amount of TyrRS·[14C]-L-TyrAMP intermediate bound to the nitrocellulose discs is quantified by scintillation counting. Comparing the TyrRSWT and TyrRS-FRSed concentrations determined by active site titration with those determined by A280 measurements indicated that the TyrRS-WT and TyrRS-FRSed enzymes were >95% active. Single Turnover Kinetic Analysis of Tyrosine Activation. Tyrosyl-tRNA synthetase undergoes a blue shift in its intrinsic

y = f0 e(−kobst ) + fc

(1)

where y is the fluorescence intensity, t is time in seconds, kobs is the observed rate constant, and fc is the background fluorescence intensity.45 Rate and dissociation constants were determined by plotting the observed rate constant (kobs), determined from eq 1, versus the substrate concentration. Data that displayed a hyperbolic dependence on substrate concentration were fit to the following equation:48

kobs =

k 3[S] Kd + [S]

(2)

where kobs and k3 are the observed rate constant (for a particular substrate concentration) and the forward rate constant for the formation of the enzyme-bound tyrosyladenylate intermediate, respectively, Kd is the equilibrium constant for dissociation of the substrate from the enzyme· substrate complex, and [S] is the initial substrate concentration. An Eadie−Hofstee transformation of eq 2 was used to determine the goodness of fit for the data:49 kobs =

−Kdkobs [S] + k 3

(3)

where kobs, Kd, and k3 are as described above. Data that displayed sigmoidal dependence with respect to substrate concentration were fit to a nonlinear Hill equation:50 kobs =

k 3[ATP]n ) (Kdn + [ATP]n )

(4)

where kobs, Kd, and k3 are as defined above and n is the Hill coefficient. To determine the goodness of fit, the data were fit to a linear transformation of eq 4:50 ⎛ kobs ⎞ log⎜ ⎟ = n log([ATP]) − log(Kd) ⎝ k 3 − kobs ⎠

(5)

where kobs/(k3-kobs) is the fraction of ATP binding sites that are occupied, and Kd and n are as defined above. Equilibrium Binding Assay. Equilibrium dialysis was performed as previously described.29,51 The number of binding sites and the dissociation constant for tyrosine (KdTyr) were determined by plotting the concentration of bound tyrosine ([TyrBound]) divided by free tyrosine ([TyrFree]) against the concentration of free tyrosine (eq 6):52 [TyrBound] [TyrFree]

=

[TyrFree] KdTyr

+

nb[E] KdTyr

(6)

where [TyrFree] is the concentration of tyrosine in the chamber B, [TyrBound] is the concentration of tyrosine in chamber A minus the concentration of tyrosine in chamber B, nb is the 1543

DOI: 10.1021/acs.biochem.5b01167 Biochemistry 2016, 55, 1541−1553

Article

Biochemistry y = y0 (1 − e−kobst )

total number of binding sites per enzyme dimer, and [E] is the concentration of tyrosyl-tRNA synthetase. Monitoring the Stability of the TyrRS·Tyr-AMP Intermediate. To synthesize the radiolabeled enzyme-bound tyrosyladenylate intermediate (TyrRS·Tyr-AMP), the enzyme (TyrRS-WT or TyrRS-FRSed) was incubated with 10 μM [14C]-L-tyrosine (482 Ci/mol) and 10 mM ATP in the presence of 100 mM Tris pH 7.8, 10 mM MgCl2, and 1 U/ mL inorganic pyrophosphatase for 20 min at 25 °C. The resulting TyrRS·[14C]Tyr-AMP intermediate was purified by size exclusion chromatography using a NAP25 column, as previously described.53 The stabilities of the enzyme-bound tyrosyl-adenylate intermediates were determined by incubating the TyrRS-WT· [14C]Tyr-AMP and the TyrRS-FRSed·[14C]Tyr-AMP complexes at 25 °C and monitoring the loss of [14C]tyrosyl-AMP from the enzyme by nitrocellulose filtration as previously described.46,54 Briefly, the TyrRS·Tyr-AMP intermediates were incubated at 25 °C in the presence of 100 mM Tris pH 7.78, 10 mM MgCl2, and aliquots were removed at regular intervals and filtered through nitrocellulose discs. The nitrocellulose discs were then washed three times with ice-cold buffer, dried, and subjected to scintillation counting to quantify the amount of enzyme-bound [14C]tyrosyl-adenylate bound to the filter. The data were fit to the following first order equation to determine the rate-constant and half-life for the loss of tyrosyl-adenylate from the enzyme, y = y0 e(−koff t )

(8)

where y is equal to the concentration of Tyr-tRNATyr at time t, y0 is the initial concentration of Tyr-tRNATyr, kobs is the observed rate constant, and t is the time in seconds. A spectrophotometric tyrosyl-tRNA synthetase assay was used to determine Km and kcat values for the TyrRS-FRSed variants.35 In this assay, the production of AMP during the tRNA aminoacylation reaction is coupled to the reduction of NAD+ by the actions of AMP deaminase and IMP dehydrogenase (Supporting Figure S1). As the production of NADH results in an increase in absorbance at 340 nm (ε340 = 6220 M−1 cm−1 ), the aminoacylation of tRNA can be followed spectrophotometrically. To prevent tRNA from being the limiting substrate in the assay, an enzyme is included that cleaves the Tyr-tRNA product, regenerating the free tRNA substrate. Cleavage of L-Tyr-tRNA is achieved by including Mycobacterium tuberculosis cyclodityrosine synthase in the assay (which converts 2 molecules of L-Tyr-tRNA to 1 molecule of cyclodityrosine and 2 molecules of tRNA), whereas cleavage of D-Tyr-tRNA is achieved by including Thermus thermophilus Dtyrosyl-tRNA deacylase (which hydrolyzes D-aminoacyltRNAs). The reaction mixture for this assay contained 50 mM Tris, pH 7.78, 10 mM KCl, 10 mM MgCl2, 0.1 mM dithiothreitol, 10 mM MgATP, 5 mM NAD+, 160 nM AMP deaminase, 640 nM IMP dehydrogenase, 2 U/mL inorganic pyrophosphatase, 5−125 nM TyrRS-WT, and variable concentrations of tyrosine and tRNATyr, depending on whether KMTyr or KMtRNA was being determined. For reactions containing L-tyrosine, the assay mix contained 8 μM cyclodityrosine synthase, while reactions with D-tyrosine contained 50 μM D-tyrosyl-tRNA deacylase. For the determination of KML‑Tyr and KMD‑Tyr, the concentration of tRNA was 5 μM, and the tyrosine concentration varied from 5 μM to 600 μM. For the determination of KMtRNA, the concentration of either L- or D-tyrosine was 500 μM, and the tRNA concentration varied from 0.1 to 10 μM. All solutions were adjusted to pH 7.0 prior to use. Assays were either 100 or 200 μL in volume (corresponding to 0.28 and 0.56 cm pathlengths, respectively) and were performed in 96-well plates at 25 °C using a Biotek Synergy 4 plate reader to monitor the change in absorbance at 340 nm. The change in absorbance was plotted against time and fit to a linear equation to determine the initial rate (vo) for each substrate concentration:35

(7)

where y is the moles of [14C]Tyr-AMP bound to the enzyme at time t, y0 is the initial number of moles of [14C]Tyr-AMP bound to the enzyme (i.e., at t = 0), koff is the rate constant for the dissociation of [14C]Tyr-AMP from the enzyme, and t is the time in seconds. Monitoring the Aminoacylation of tRNATyr. The steadystate aminoacylation of tRNATyr was monitored using two approaches: (1) the incorporation of [14C]-L- or D-tyrosine into the tyrosyl-tRNA product by tyrosyl-tRNA synthetase was monitored using a nitrocellulose filter assay, and (2) the formation of the tyrosyl-tRNA product was monitored by following the concomitant release of AMP using a continuous spectrophotometric tyrosyl-tRNA synthetase assay.35,38 To demonstrate that the TyrRS-FRSed variant is active, incorporation of [14C]tyrosine into the tyrosyl-tRNATyr product was monitored by incubating the enzyme (TyrRS-WT or TyrRSFRSed) with 10 mM ATP, 100 mM Tris, pH 7.8, 10 mM MgCl2, 2 U/mL inorganic pyrophosphatase, and either 10 μM [14C]-L-tyrosine (482 Ci/mol) or 25 μM [14C]-D-tyrosine (55 Ci/mol) and various concentrations of tRNATyr (1−8 μM). Enzyme concentrations were 10−50 nM for TyrRS-WT and 100−500 nM for TyrRS-FRSed. The tRNA aminoacylation reactions were incubated at 25 °C, and 10 μL aliquots were removed and quenched at regular intervals by the addition to 3 mL of cold 5% trichloroacetic acid. The aliquots were then filtered through nitrocellulose discs to separate the [14C]tyrosyl-tRNATyr product from free [14C]tyrosine, and the filters were washed three times with cold 3 mL 5% trichloroacetic acid, dried, and subjected to scintillation counting to quantify the amount of [14C]tyrosyl-tRNA bound to the filter. The data were plotted (cpm versus time) and fit to a first order rate equation:55

A340 = vot + C

(9)

where A340 is the absorbance at 340 nm at time “t”, vo is the initial rate, and C is the initial absorbance at t = 0. After subtracting out the background rate (i.e., the initial rate in the absence of enzyme), the initial rates were plotted against substrate concentration and the data were fit to the Michaelis− Menten equation to determine the KM and Vmax values:56,57 v0 =

Vmax[S] KM + [S]

(10)

where vo represents the initial rate, [S] represents the initial substrate concentration, KM represents the Michaelis constant for the substrate, and Vmax is the maximal rate of the reaction.56 The kcat values were determined from Vmax using the eq 11:56,57 Vmax = kcat[E] 1544

(11) DOI: 10.1021/acs.biochem.5b01167 Biochemistry 2016, 55, 1541−1553

Article

Biochemistry where kcat is the turnover number, [E] is the total enzyme concentration, and Vmax is defined above. Monitoring Transediting of the Tyrosyl-tRNATyr Product. Trans-editing of the L- and D-tyrosyl-tRNA products was monitored by enzymatically synthesizing the [14C]-L- or Dtyrosyl-tRNA product and monitoring its hydrolysis via a nitrocellulose filter binding assay. [14C]Tyrosyl-tRNATyr was synthesized by incubating 100 nM TyrRS-WT, 10 mM MgATP, 5 μM tRNATyr and either 10 μM [14C]-L-tyrosine or 25 μM [14C]-D-tyrosine in the presence of 100 mM Tris, pH 7.8, 10 mM MgCl2, and 2 U/mL inorganic pyrophosphatase at 25 °C for either 20 min (L-tyrosine) or 40 min (D-tyrosine). The [14C]tyrosyl-tRNA product was purified by two extractions with phenol:chloroform:isoamylalcohol (50:49:1), followed by the addition of 0.5 vol of 7.5 M ammonium acetate and 3 vol of ethanol to precipitate the [14C]tyrosyl-tRNA. Dried pellets were resuspended in 100 mM HEPES, 10 mM MgCl2, pH 7.5, and 5 μL aliquots were removed, spotted onto nitrocellulose discs, and subjected to scintillation counting to determine the concentration of [14C]tyrosyl-tRNATyr. For the trans-editing assay, [14C]tyrosyl-tRNA (2−5 μM) was incubated with 250 nM TyrRS-WT or TyrRS-FRSed in the presence of 100 mM HEPES, pH 7.5, and 10 mM MgCl2. Ten microliter aliquots were removed and quenched by the addition to 3 mL of 5% trichloroacetic acid, filtered through nitrocellulose discs to separate [14C]tyrosyl-tRNATyr from [14C]tyrosine, and subjected to scintillation counting to quantify the amount of [14C]tyrosyl-tRNATyr bound to the nitrocellulose discs. The data were plotted (cpm versus time) and fit to a first-order decay equation with floating end point:58 y = y0 e−k1t + y∞

tion counter was calibrated using commercial standards (Beckman Coulter), and quench curves for the dual label counting were setup by spotting [14C]-L-tyrosine and [3H]-Dtyrosine onto nitrocellulose discs, adding 5.5 mL Biosafe II scintillation cocktail to 7 mL scintillation vials, and adding increasing amounts of chloroform to the vials.59 Counting efficiencies for [3H]-D-tyrosine were near 40% and efficiencies for [14C]-L-tyrosine were greater than 85%, compared to the calculated disintegrations per minute. After normalizing for counting efficiency, the data were plotted as [D-Tyr-tRNA]/[LTyr-tRNA] vs the initial concentration of D-tyrosine and fit to a linear equation.

■

RESULTS The Phenylalanyl-tRNA Synthetase Editing Domain Is Stereospecific. Saccharomyces cerevisiae phenylalanyl-tRNA synthetase contains an editing domain in its β-subunit that hydrolyzes the Tyr-tRNAPhe misacylation product.60,61 A transediting assay was performed to determine the enantioselectivity of this editing domain. In this assay, either [14C]-L- or [14C]DTyr-tRNATyr is incubated in the presence or absence of the phenylalanyl-tRNA synthetase editing domain. Hydrolysis of Land D-Tyr-tRNATyr is monitored by capturing the remaining [14C]Tyr-tRNATyr on nitrocellulose discs and quantifying the amount of radioactivity bound to the disc by scintillation counting. S. cerevisiae phenylalanyl-tRNA synthetase was found to hydrolyze L-Tyr-tRNATyr with a rate constant of 0.002 s−1 (Figure 1A). In contrast, the activity of S. cerevisiae phenylalanyl-tRNA synthetase toward D-Tyr-tRNATyr is at least 100fold less than its activity toward L-Tyr-tRNATyr (Figure 1B). These results demonstrate that the phenylalanyl-tRNA synthetase editing domain is stereospecific, as it only hydrolyzes L-tyrosyl-tRNA. In addition, the results confirm a previous observation by Yokoyama and colleagues that the editing domain recognizes and hydrolyzes both L-Tyr-tRNAPhe and LTyr-tRNATyr.30 Tyrosyl-tRNA synthetase catalyzes the aminoacylation of tRNATyr by both L- and D-tyrosine. The observation that the phenylalanyl-tRNA synthetase editing domain selectively hydrolyzes L-Tyr-tRNA suggests that the enantioselectivity of tyrosyl-tRNA synthetase can be altered by inserting the editing domain into tyrosyl-tRNA synthetase. A similar approach was used by Yokoyama and colleagues to reduce the formation of Ltyrosyl-tRNATyr by a tyrosyl-tRNA synthetase variant that aminoacylates tRNATyr with 3-iodo-L-tyrosine.30 To determine whether the enantioselectivity of tyrosyl-tRNA synthetase can be altered by inserting an editing domain, the P. horikoshii phenylalanyl-tRNA synthetase editing domain (residues 83− 275 in the β subunit) was inserted between residues 161 and 162 in the CP1 domain of G. stearothermophilus tyrosyl-tRNA synthetase (Supporting Figure S2). The resulting protein is designated TyrRS-FRSed. Expression of TyrRS-FRSed and the wild-type G. stearothermophilus tyrosyl-tRNA synthetase (TyrRS-WT) enzymes results in proteins that are >95% pure based on SDS-PAGE. Typical yields of the TyrRS-FRSed variant (2−5 mg/L of cell culture) are ∼10-fold lower than those for wild type tyrosyl-tRNA synthetase, suggesting that the TyrRS-FRSed variant may be less stable than the wild type enzyme. Tyrosyl-tRNA synthetase is a homodimeric enzyme that exhibits an extreme form of negative cooperativity, known as “half-of-the-sites reactivity” in which only one molecule of tyrosine binds and is activated per dimer. The amount of

(12)

where y is the concentration of [14C]-labeled tyrosyl-tRNATyr at time t, y0 is the initial concentration of [14C]-labeled tyrosyltRNATyr (i.e., at t = 0), y∞ is the final concentration of [14C]labeled [TyrRS-tRNA] (i.e., at an infinite time point), k1 is the rate of hydrolysis, and t is the time in seconds. The standard error in the rate constants determined for the wild type tyrosyltRNA synthetase, S. cerevisiae phenylalanyl-tRNA synthetase editing domain, and TyrRS-FRSed variant was used to estimate the lower limit of the assay. Monitoring the Competition between L- and D-Tyrosine. A competition assay was used to quantify the effect that introducing the phenylalanyl-tRNA synthetase editing domain into tyrosyl-tRNA synthetase has on the enantioselectivity of the enzyme. Competition assays contained 10 mM MgATP, 100 mM Tris pH 7.78, 10 mM MgCl2, 1 U/mL inorganic pyrophosphatase, 10 μM tRNA, 25 nM TyrRS-WT, or 250 nM TyrRS-FRSed, 30 μM [14C]-L-tyrosine, and variable concentrations of [3H]D-tyrosine (0−120 μM). Assays were incubated at 25 °C for 7.5 min, 20 μL aliquots were removed, and the aliquots were quenched by the addition of 3 mL ice cold 5% trichloroacetic acid. The 7.5 min time point was selected based on the observation that this time point was within the initial linear phase of the reaction for both the TyrRS-WT and TyrRSFRSed enzymes. Following quenching of the reaction, the aliquots were filtered through nitrocellulose discs that had been presoaked in 5% trichloroacetic acid. The nitrocellulose discs were washed three times with 3 mL of ice-cold 5% trichloroacetic acid, dried, and subjected to scintillation counting to quantify the amount of [14C]-L-tyrosyl-tRNA and [3H]-D-tyrosyl-tRNA present. The Beckman LS-6500 scintilla1545

DOI: 10.1021/acs.biochem.5b01167 Biochemistry 2016, 55, 1541−1553

Article

Biochemistry

Figure 1. The phenylalanyl-tRNA synthetase editing domain is stereospecific. The ability of phenylalanyl-tRNA synthetase to hydrolyze L- and D-Tyr-tRNATyr was quantified using a trans-editing assay. Typical curves for the hydrolysis of [14C]-L-Tyr-tRNATyr (panel A) or [14C]-L-Tyr-tRNATyr (panel B) in the presence (circles) or absence (squares) of S. cerevisiae phenylalanyl-tRNA synthetase are shown.

Figure 2. The TyrRS-FRSed variant aminoacylates tRNA with both Land D-tyrosine. To determine whether tyrosyl-tRNA synthetase retains activity after insertion of the phenylalanyl-tRNA synthetase editing domain, the incorporation of [14C]tyrosine into the Tyr-tRNA product was monitored using a nitrocellulose filter binding assay. To compensate for the decreased specific activity of the TyrRS-FRSed variant, its concentration is 10-fold higher than that of the wild-type tyrosyl-tRNA synthetase in these assays. Typical curves for the aminoacylation of tRNATyr by [14C]-L-tyrosine (panel A) or [14C]-Dtyrosine (panel B) in the presence of either wild type tyrosyl-tRNA synthetase (squares) or the TyrRS-FRSed variant (circles) are shown.

tyrosine bound to the tyrosyl-tRNA synthetase dimer can be determined using an active site titration assay.44 In this assay, tyrosyl-tRNA synthetase is incubated with [14C]tyrosine and ATP, forming the TyrRS·Tyr-AMP intermediate. Comparing the amount of [14C]tyrosine incorporated into the TyrRSFRSed·Tyr-AMP intermediate with the amount of TyrRSFRSed present in the assay indicates that, like the wild type enzyme, the TyrRS-FRSed variant displays half-of-the-sites reactivity with respect to the enzyme-bound tyrosyl-adenylate intermediate (Supporting Figure S3). To determine whether tyrosyl-tRNA synthetase is active after insertion of the phenylalanyl-tRNA synthetase editing domain, the ability of the TyrRS-FRSed variant to catalyze the formation of both L- and D-tyrosyl-tRNA was analyzed by monitoring the incorporation of either [14C]-L- or [14C]-D-tyrosine into tyrosyltRNATyr (Figure 2, panels A and B, respectively). In this assay, the [14C]Tyr-tRNATyr product is separated from [14C]tyrosine by nitrocellulose filtration, and the amount of [14C]TyrtRNATyr bound to the nitrocellulose disc is quantified by scintillation counting. As the specific activity of the TyrRSFRSed variant is ∼10-fold lower than that of the wild type tyrosyl-tRNA synthetase, 10-fold higher concentrations of TyrRS-FRSed were used in the assay. Despite this 10-fold increase in the TyrRS-FRSed concentration, the final concentration of L-Tyr-tRNA is approximately 50% lower for the TyrRS-FRSed variant than it is for the wild type enzyme. In contrast, when tRNATyr is aminoacylated by D-tyrosine, the final concentration of D-Tyr-tRNA is similar for the TyrRS-FRSed

and TyrRS-WT variants. These observations are consistent with the hypothesis that the editing domain in the TyrRS-FRSed variant hydrolyzes L-tyrosyl-tRNATyr, but not D-tyrosyltRNATyr. To determine the stereospecificity of the P. horikoshii phenylalanyl-tRNA synthetase editing domain, a trans-editing assay was used to monitor hydrolysis of L- and D-Tyr-tRNATyr by the TyrRS-FRSed variant. This assay indicates that the editing domain in the TyrRS-FRSed variant hydrolyzes L-TyrtRNATyr, but not D-Tyr-tRNATyr (Figure 3, panels A and B, respectively). Analysis of trans-editing activity in an editingdeficient variant of TyrRS-FRSed (TyrRS-FRSed-N217A) confirms that hydrolysis of L-Tyr-tRNATyr is due to the inserted phenylalanyl-tRNA synthetase editing domain (Figure 3). Insertion of the Phenylalanyl-tRNA Synthetase Editing Domain into Tyrosyl-tRNA Synthetase Reduces Its Affinity for L- and D-Tyrosine under Single Turnover Conditions. Steady state aminoacylation assays monitoring the incorporation of radiolabeled tyrosine into tRNA indicate that the TyrRS-FRSed variant is less active than wild-type tyrosyltRNA synthetase (Figure 2). To determine how insertion of the editing domain affects the catalytic activity of tyrosyl-tRNA 1546

DOI: 10.1021/acs.biochem.5b01167 Biochemistry 2016, 55, 1541−1553

Article

Biochemistry

determine the rate and dissociation constants for each step in the reaction mechanism.62 To determine the equilibrium constant for the dissociation of tyrosine from the TyrRS·Tyr complex (KdTyr) the assay was performed under conditions where ATP is not bound to the enzyme prior to tyrosine binding (i.e., [ATP] ≤ 0.1KdATP). Similarly, the equilibrium constant for the dissociation of tyrosine from the TyrRS·Tyr·ATP complex is determined by performing the assay under conditions where the enzyme is saturated with ATP. As high concentrations of ATP (>10 mM) are inhibitory for tyrosyl-tRNA synthetase, we have used 10 mM ATP to determine the K′dTyr ([ATP] = 2.5KdATP, which is equivalent to 70% saturation of the binding site). This methodology is consistent with previously published kinetic analyses of tyrosyl-tRNA synthetase.45,53,54 Single-turnover kinetic analyses indicate that the editing domain reduces the affinity of tyrosyl-tRNA synthetase for L- and D-tyrosine by 10and 3-fold, respectively (Supporting Figure S4, Table 1). Similarly, insertion of the editing domain reduces the affinity of the TyrRS·Tyr·ATP complex for L- and D-tyrosine by 5- and 1.4-fold, respectively (Supporting Figure S5, Table 1). The equilibrium constants for the dissociation of ATP from the TyrRS·ATP (KdATP) and the TyrRS·Tyr·ATP complexes (K′dATP) are determined by performing the single turnover kinetic assay under conditions where [Tyr] ≤ 0.1KdTyr and [Tyr] ≥ 10KdTyr, respectively. Insertion of the editing domain has little effect on the ATP binding affinity of either the TyrRS· ATP or the TyrRS·ATP·L-Tyr complexes (Supporting Figures S6 and S7, Table 1). It has previously been observed that in the presence of saturating concentrations of D-tyrosine, G. stearothermophilus tyrosyl-tRNA synthetase displays cooperative kinetics with respect to the binding of ATP.63 It is postulated that the cooperative binding of ATP is an intrinsic property of the enzyme, but that it is not normally observed due to half-ofthe-sites reactivity, which masks the binding of ATP to the inactive subunit. Binding of D-tyrosine, however, decreases the ATP binding affinity of the active subunit and allows ATP to bind initially to the inactive subunit, revealing the intrinsic cooperativity of the enzyme.63 Insertion of the phenylalanyltRNA synthetase editing domain does not perturb the cooperative binding of ATP in the presence of D-tyrosine (Supporting Figure S7C,D, Table 1). In theory, the forward rate constant for the tyrosine activation reaction, k3, can be determined by performing the single turnover kinetic assay at saturating concentrations of either ATP or tyrosine. In practice, however, the decreased affinity of the active subunit for ATP prevents saturating concentrations of ATP from being used when D-tyrosine is bound to the enzyme. As a result, the k3 values were determined using saturating concentrations of L- and D-tyrosine. Insertion of the phenylalanyl-tRNA synthetase editing domain

Figure 3. The phenylalanyl-tRNA synthetase editing domain in the TyrRS-FRSed variant is stereospecific. The stereospecificity of the editing domain in the TyrRS-FRSed variant was determined using a trans-editing assay. Typical curves for the hydrolysis of either [14C]-LTyr-tRNA (panel A) or [14C]-D-Tyr-tRNA (panel B) in the presence of wild type tyrosyl-tRNA synthetase (open circle), the TyrRS-FRSed variant (filled triangle), the editing defective TyrRS-FRSed-N217A variant (filled diamond), or in the absence of tyrosyl-tRNA synthetase (open square) are shown.

synthetase, stopped-flow fluorescence spectroscopy was used to monitor the single turnover kinetics of the TyrRS-FRSed variant. This method takes advantage of the observation that formation of the TyrRS·Tyr-AMP intermediate is accompanied by a blue shift in the intrinsic fluorescence of the enzyme. As this change in intrinsic fluorescence correlates with formation of the transition state for the tyrosine activation step, rate and dissociation constants for the activation of L- and D-tyrosine can be determined by monitoring the change in fluorescence above 320 nm. Furthermore, since the reaction is performed in the absence of tRNA and the tyrosyl-adenylate intermediate does not dissociate during the time course of the reaction, only a single turnover of the enzyme occurs. This makes it possible to

Table 1. Single Turnover Kinetic Data for the Tyrosyl-tRNA Synthetase Variantsa TyrRS-WT

L-Tyr D-Tyr

TyrRS-FRSed

L-Tyr D-Tyr

KdTyr (μM)

K′dTyr (μM)

KdATP (mM)

K′dATP (mM)

2 (±3) 62 (±6) 380 (±60) 190 (±30)

43 (±9) 150 (±10) 220 (±20) 210 (±30)

10 (±2) 14 (±3) 18 (±2) 9 (±2)

2.0 (±0.2) 6 (±2) 3.8 (±0.5) 12.6 (±0.7)

k3 (s−1) 25 19 17 17

(±1) (±4) (±1) (±1)

n NA 2.0 (±0.3) NA 2.4 (±0.3)

a

KdTyr and K′dTyr represent the tyrosine dissociation constants for the TyrRS·Tyr and TyrRS·Tyr·ATP complexes, respectively, KdATP and K′dATP represent the ATP dissociation constants for the TyrRS·ATP and TyrRS·Tyr·ATP complexes, respectively, k3 represents the forward rate constant for the formation of the TyrRS·Tyr-AMP complex, n represents the Hill coefficient, and NA indicates “not applicable”. 1547

DOI: 10.1021/acs.biochem.5b01167 Biochemistry 2016, 55, 1541−1553

Article

Biochemistry into tyrosyl-tRNA synthetase was found to have only a minimal effect on the forward rate constant for both L- and D-tyrosine activation (Supporting Figure S5B,D, Table 1). To confirm the above results, the effect that inserting the phenylalanyl-tRNA synthetase editing domain has on the tyrosine binding affinity of tyrosyl-tRNA synthetase was determined using equilibrium dialysis. While wild-type tyrosyl-tRNA synthetase showed L- and D-tyrosine binding affinities similar to previously reported values, binding of L- and D-tyrosine to the TyrRS-FRSed variant could not be detected by equilibrium dialysis (Supporting Figure S8). The inability to detect binding of L- and D-tyrosine by equilibrium dialysis implies that KdD-Tyr > 100 μM, which is consistent with the KdD-Tyr determined for the TyrRS-FRSed variant in the single turnover kinetic assays. Inserting the Phenylalanyl-tRNA Synthetase Editing Domain into Tyrosyl-tRNA Synthetase Decreases Its Affinity for the Tyrosyl-Adenylate Intermediate. Attempts to use single turnover kinetics to monitor transfer of the tyrosyl moiety to tRNA proved to be problematic due to difficulties in isolating sufficient quantities of the TyrRS-FRSed· Tyr-AMP complex, suggesting that the TyrRS-FRSed variant has a lower affinity for the tyrosyl-adenylate intermediate than the wild type enzyme. To test the hypothesis that inserting the phenylalanyl-tRNA synthetase editing domain decreases the affinity of tyrosyl-tRNA synthetase for the tyrosyl-adenylate intermediate, the TyrRS·Tyr-AMP and TyrRS-FRSed·TyrAMP complexes were formed with [14C]-L-tyrosine and separated from free [14C]-L-tyrosine using a NAP25 column. Loss of [14C]Tyr-AMP from the TyrRS·Tyr-AMP and TyrRSFRSed·Tyr-AMP intermediates was monitored using a nitrocellulose filter binding assay. In this assay, the TyrRS·[14C]TyrAMP intermediate is incubated at 25 °C, and aliquots are removed at various time points and filtered through nitrocellulose discs to capture the TyrRS·[14C]Tyr-AMP intermediate. The TyrRS·[14C]-L-Tyr-AMP intermediate was found to have a half-life of ∼150 min at 25 °C (koff = 8 × 10−5 s−1), consistent with published results for the untagged enzyme.64 In contrast, the TyrRS-FRSed·[14C]-L-Tyr-AMP intermediate has a half-life of ∼20 min (koff = 6 × 10−4 s−1) confirming that the TyrRS-FRSed variant has a decreased affinity for the tyrosyladenylate intermediate (Figure 4). Inserting the Phenylalanyl-tRNA Synthetase Editing Domain Reduces the Catalytic Rate of Tyrosyl-tRNA Synthetase. The decreased stability of the TyrRS-FRSed·TyrAMP intermediate prevents the use of single-turnover kinetics to monitor the second step of the tRNA aminoacylation reaction (i.e., transfer of the tyrosyl moiety to tRNA). As an alternate approach, a spectrophotometric steady-state tyrosyltRNA synthetase assay was used to determine whether insertion of the editing domain affects the binding of the Ltyrosine, D-tyrosine, or tRNA substrates.35 This assay takes advantage of the observation that AMP is released during the tRNA aminoacylation reaction. The release of AMP is coupled to the production of NADH via AMP deaminase and IMP dehydrogenase, allowing the reaction to be monitored by following the increase in absorbance at 340 nm (Supporting Figure S1). In addition, to prevent tRNA from being the limiting substrate in the assay, the Tyr-tRNA product is cleaved by either cyclodityrosine synthase (for L-Tyr-tRNA) or Dtyrosyl-tRNA deacylase (for D-Tyr-tRNA), regenerating the tRNA substrate. It should be noted that since the Tyr-tRNA product is cleaved in this assay, only the activity of the synthetic

Figure 4. Dissociation of the tyrosyl-adenylate intermediate from wild type tyrosyl-tRNA synthetase and the TyrRS-FRSed variant. The rate for the dissociation of tyrosyl-adenylate from the TyrRS·Tyr-AMP and TyrRS-FRSed·Tyr-AMP complexes was quantified by monitoring the loss of [14C]Tyr-AMP from the enzyme. Typical curves for the release of L-Tyr-AMP from TyrRS·Tyr-AMP (squares) and TyrRS-FRSed· Tyr-AMP (circles) are shown. The release of D-tyrosyl-adenylate was not determined as the TyrRS-FRSed·[14C]-D-Tyr-AMP complex could not be isolated.

site is monitored and not the effect that the editing domain has on the final product. As a result, the presence or absence of the editing domain will have no effect on the kcat and Km values obtained from the spectrophotometric assay. Although single turnover kinetic analyses indicate that insertion of the phenylalanyl-tRNA synthetase editing domain decreases the binding affinity for L- and D-tyrosine by 5- and 1.4-fold, respectively, this effect is not observed under steady state conditions (Supporting Figure S9, Table 2). However, TyrRS-FRSed does show 20- and 10-fold decreases in the kcat values for L- and D-tyrosine, respectively, compared to the wildtype enzyme (Table 2). This reduced activity is independent of the catalytic activity of the inserted editing domain, as steadystate kinetics for the editing defective N217A variant of TyrRSFRSed are similar to those of TyrRS-FRSed. Insertion of the phenylalanyl-tRNA synthetase editing domain also decreases the affinity of the TyrRS·Tyr-AMP complex for tRNA by 2-fold when L-tyrosine is bound, but not when D-tyrosine is bound (Supporting Figure S10, Table 2). Inserting the Phenylalanyl-tRNA Synthetase Editing Domain Alters the Stereospecificity of Tyrosyl-tRNA Synthetase. To determine the effect that inserting the phenylalanyl-tRNA synthetase editing domain has on the enantioselectivity of tyrosyl-tRNA synthetase, the relative abilities of L- and D-tyrosine to aminoacylate tRNATyr were determined using a competition assay. In this assay, [14C]-Ltyrosine, [3H]-D-tyrosine, ATP, and tRNATyr are incubated with either wild-type tyrosyl-tRNA synthetase or the TyrRS-FRSed variant. After 7.5 min, aliquots are removed, quenched, and the radiolabeled tyrosyl-tRNA products are isolated by nitrocellulose filtration, followed by scintillation counting to quantify the amounts of [14C]-L-tyrosyl-tRNATyr and [3H]-D-tyrosyltRNATyr present. The competition assay was performed using 30 μM Ltyrosine and varying concentrations of D-tyrosine. At equimolar concentrations of L- and D-tyrosine (i.e., 30 μM), the ratio of DTyr-tRNA to L-Tyr-tRNA produced by the wild-type tyrosyltRNA synthetase is ∼1:10 (Figure 5). This ratio increases to ∼1:5 when the TyrRS-FRSed variant is used to catalyze the tRNA aminoacylation reaction. Increasing the concentration of 1548

DOI: 10.1021/acs.biochem.5b01167 Biochemistry 2016, 55, 1541−1553

Article

Biochemistry Table 2. Steady-State Kinetic Data for the Tyrosyl-tRNA Synthetase Variants KMTyr (μM) TyrRS-WT

a

L-Tyr D-Tyr

TyrRS-FRSed WT

L-Tyr D-Tyr

TyrRS-FRSed-N217A

L-Tyr D-Tyr

a

24 23 30 33 20 45

(±3) (±6) (±10) (±3) (±5) (±5)

KMtRNA (μM)

kcat (s−1)

0.94 (±0.02) 0.33 (±0.06) 1.8 (±0.1) 0.24 (±0.04) 2.7 (±0.4) 0.37 (±0.04)

2.8 (±0.6) 0.16 (±0.04) 0.17 (±0.02) 0.032 (±0.002) 0.10 (±0.03) 0.039 (±0.008)

TyrRS-WT numbers are taken from ref 35.

perturbs the tyrosyl-tRNA synthetase active site, but that the binding of tRNA counteracts this effect, stabilizing an active site conformation that binds both L- and D-tyrosine with high affinity. Alternatively, it may be that the process of undergoing the initial round of catalysis stabilizes the tyrosyl-tRNA synthetase active site in a conformation that binds L- and Dtyrosine with high affinity. In either case, it is clear that under multiple turnover conditions, insertion of the phenylalanyltRNA synthetase editing domain does not significantly affect the binding of L- or D-tyrosine. The observation that insertion of the phenylalanyl-tRNA synthetase editing domain affects the initial binding of the tyrosine substrate, as well as the enzyme turnover rate implies that the synthetic site in tyrosyl-tRNA synthetase is affected by distal changes in the CP1 domain. In this regard, tyrosyl-tRNA synthetase appears to be similar to another class I aminoacyltRNA synthetase, Mycoplasma mobile leucyl-tRNA synthetase, which lacks a CP1 editing domain. Insertion of the CP1 editing domain from E. coli leucyl-, isoleucyl-, or valyl-tRNA synthetase decreases the binding affinity for both cognate and noncognate amino acids (leucine, isoleucine, valine, methionine), decreases the enzyme turnover rate, and increases the specificity of the synthetic site. On the basis of these observations, Boniecki and Martinis proposed that, like tyrosyl-tRNA synthetase, the synthetic site in M. mobile leucyl-tRNA synthetase is affected by distal changes in the CP1 domain of the enzyme.65 Editing by aminoacyl-tRNA synthetases falls into two main categories: (1) hydrolysis of the noncognate aminoacyladenylate intermediate (pretransfer editing), and (2) hydrolysis of the noncognate aminoacyl-tRNA product (post-transfer editing).66−69 To be effective, the rate of editing must be comparable to, or exceed, the rate for synthesis of the noncognate aminoacyl-AMP intermediate (for pretransfer editing) or noncognate aminoacyl-tRNA product (for posttransfer editing). This is exemplified by comparing editing by the valyl- and isoleucyl-tRNA synthetases, both of which have post-transfer editing domains. In valyl-tRNA synthetase, the rate for transfer of the noncognate aminoacyl moiety to the 3′ end of tRNA is fast (ktrans = 55 s−1).70 Since H2O is unable to compete with the 2′ OH of A76 in tRNA for nucleophilic attack on the aminoacyl-adenylate intermediate, the rate of pretransfer editing is low in this enzyme. As a result, valyl-tRNA synthetase relies on post-transfer editing to remove the noncognate aminoacyl-tRNA products. In contrast, the rate for transfer of the noncognate aminoacyl moiety to tRNA is ∼100-fold slower in isoleucyl-tRNA synthetase (ktrans = 0.4 s−1), allowing H2O to compete with the 2′ OH of A76. This allows pretransfer editing to play a significant role in maintaining the fidelity of isoleucyl-tRNA synthetase.70 In class I aminoacyl-tRNA synthetases that have post-transfer editing domains (i.e., isoleucyl-, leucyl-, and valyl-tRNA synthetases), translocation of the 3′ end of the noncognate

Figure 5. Competition between L- and D-tyrosine for aminoacylation of tRNA by TyrRS-WT and TyrRS-FRSed. Competition assays were performed in the presence of 30 μM L-tyrosine and varying concentrations of D-tyrosine. The ratio of [D-Tyr-tRNA]/[L-TyrtRNA] produced after 7.5 min is shown for the wild-type tyrosyl-tRNA synthetase (squares), TyrRS-FRSed (circles), and TyrRS-FRSedN217A (diamonds). Error bars indicate standard error values.

to 120 μM (i.e., 4-fold higher than the concentration of L-tyrosine) results in a final D-tyrosyl-tRNA:L-tyrosyl-tRNA ratio of 0.6. D-tyrosine

■

DISCUSSION The goal of the research presented here is to test the hypothesis that insertion of an editing domain into tyrosyl-tRNA synthetase will alter its enantioselectivity. Toward this end, the P. horikoshii phenylalanyl-tRNA synthetase editing domain (amino acids 83−275 in the phenylalanyl-tRNA synthetase βsubunit) was inserted between Gly 161 and Ile 162 in the CP1 domain of tyrosyl-tRNA synthetase. During the course of these investigations, we have shown that the phenylalanyl-tRNA synthetase editing domain is stereospecific, hydrolyzing only LTyr-tRNA and not D-Tyr-tRNA. Furthermore, we have confirmed the observation of Yokoyama and colleagues that recognition of the tRNA moiety by the phenylalanyl-tRNA synthetase editing domain is relatively nonspecific, as it is able to hydrolyze both L-Tyr-tRNAPhe and L-Tyr-tRNATyr.30 We have also shown that inserting the phenylalanyl-tRNA synthetase editing domain into the tyrosyl-tRNA synthetase CP1 domain does not significantly alter the forward rate constant for the activation of tyrosine (k3), the binding of ATP, the binding of tRNA, or half-of-the-sites reactivity. Surprisingly, insertion of the phenylalanyl-tRNA synthetase editing domain decreased the affinity of tyrosyl-tRNA synthetase for both Land D-tyrosine under single turnover conditions and in equilibrium dialysis experiments, but not under multiple turnover conditions. There are two plausible explanations for this discrepancy. First, tRNA was not present during either the single turnover kinetic or equilibrium dialysis experiments. It is possible that the phenylalanyl-tRNA synthetase editing domain 1549

DOI: 10.1021/acs.biochem.5b01167 Biochemistry 2016, 55, 1541−1553

Article

Biochemistry

colleagues have identified six alanine substitutions that increase the activity of the phenylalanyl-tRNA synthetase editing domain, raising the possibility that the enantioselectivity of the TyrRS-FRSed variant can be increased.75 Lastly, the specificity of the synthetic site can be altered to increase the tRNA aminoacylation rate for D-tyrosine and/or decrease the tRNA aminoacylation rate for L-tyrosine. Simonson et al. have used this last approach to engineer a tyrosyl-tRNA synthetase variant that preferentially aminoacylates tRNATyr with Dtyrosine (based on kcat/Km values).76 Unfortunately, kcat is decreased at least 20 000-fold in this variant, making it unsuitable for incorporating D-tyrosine into proteins.

aminoacyl-tRNA product appears to occur without dissociation of the aminoacyl-tRNA from the enzyme (reviewed in ref 71). In contrast, post-transfer editing by phenylalanyl-tRNA synthetase involves dissociation of the noncognate aminoacyltRNA from the active site and subsequent rebinding to the editing domain.72 This may explain why the phenylalanyl-tRNA synthetase editing domain is active independently of the remainder of the phenylalanyl-tRNA synthetase protein. Yokoyama and colleagues originally inserted the phenylalanyltRNA synthetase editing domain at three locations in iodotyrosyl-tRNA synthetase: (1) at the amino-terminus, (2) at the carboxyl-terminus, and (3) in the CP1 domain. The observation that hydrolysis of L-Tyr-tRNA is most efficient when the phenylalanyl-tRNA synthetase editing domain is inserted into the tyrosyl-tRNA synthetase CP1 domain was originally attributed to the 40−50 Å spacing between the editing and synthetic sites.30 It is now apparent that much, if not all, of the increased efficiency of hydrolysis in this variant is due to a decrease in the activity of tyrosyl-tRNA synthetase. This allows the editing domain to compete with the synthetic site and provides a good illustration of the how the balance between synthesis and editing is critical to the fidelity of the enzyme. Under normal conditions, misacylation of tRNA occurs at a much lower frequency than aminoacylation by the cognate amino acid. As a result, post-transfer editing only needs to remove relatively minor contaminants. Even in the case of the engineered iodotyrosyl-tRNA synthetase that uses a phenylalanyl-tRNA synthetase editing domain to remove L-Tyr-tRNA, the activation rate for 3-iodo-L-tyrosine is 10-fold higher than the activation rate for L-tyrosine.30,73 Altering the enantioselectivity of tyrosyl-tRNA synthetase presents a more difficult problem, as the product being removed, L-tyrosyl-tRNA, is actually the favored product of the reaction. Despite this, by introducing the phenylalanyl-tRNA synthetase editing domain, we have succeeded in engineering a tyrosyl-tRNA synthetase variant that results in 40% of the tRNATyr being aminoacylated by D-tyrosine (albeit at a D-tyrosine:L-tyrosine ratio of 4:1). This achievement is primarily due to (1) a relatively small (30fold) difference between the rate at which tyrosyl-tRNA synthetase catalyzes the aminoacylation of tRNA by D- and Ltyrosine, and (2) the ability of the phenylalanyl-tRNA synthetase editing domain to compete with the tyrosyl-tRNA synthetase synthetic site. There are several approaches that can be taken to increase the ability of the TyrRS-FRSed variant to preferentially aminoacylate tRNA with D-tyrosine. First, the concentration of D-tyrosine relative to that of L-tyrosine can be increased. As the competition assay showed, even a D-tyrosine:L-tyrosine ratio of 4:1 is sufficient to significantly affect the enantioselectivity of the TyrRS-FRSed variant. This approach is limited by both the necessity of having L-tyrosine in the growth media and the relatively low solubility of tyrosine in water (2.8 mM at 25 °C, pH 7.0), which prevents high concentrations from being used.74 A second approach is to decrease the catalytic activity of the synthetic site in tyrosyl-tRNA synthetase, thereby increasing the ability of the post-transfer editing domain to compete with the synthesis of L-tyrosyl-tRNA. The limitation of this approach is that decreasing the activity of the synthetic site will decrease the rate at which tRNA is aminoacylated by both L- and D-tyrosine (although this can be compensated for by overexpressing the TyrRS-FRSed variant). A third approach is to increase the activity of the editing site. In this regard, Yokoyama and

■

CONCLUDING REMARKS We have altered the enantioselectivity of tyrosyl-tRNA synthetase by inserting the post-transfer editing domain from phenylalanyl-tRNA synthetase into the CP1 domain of tyrosyltRNA synthetase. As the phenylalanyl-tRNA synthetase editing domain is stereospecific, it selectively hydrolyzes L-Tyr-tRNA but not D-Tyr-tRNA. Introducing this editing domain into tyrosyl-tRNA synthetase reduces the activity of the synthetic site, allowing post-transfer editing to compete with the synthesis of L-Tyr-tRNA by the tyrosyl-tRNA synthetase active site.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b01167.

■

Supporting figure legends and Supporting Figures S1− S10 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: efi[email protected]. Phone: (318) 675-7779. Fax: (318) 675-5180. Present Address ∥

(C.J.R.) Department of Chemistry, University of Virginia, Charlottesville, VA 22904. Funding

This research was supported by grants from the Biomedical Research Foundation of Northwest Louisiana and the Stiles Fund of the Louisiana State University Health Sciences Center in Shreveport. Notes

The authors declare the following competing financial interest(s): A provisional patent application based on the experiments described in this paper has been filed.

■

ACKNOWLEDGMENTS The authors gratefully acknowledge Professor Michael Ibba (The Ohio State University) for his generous donation of the expression clone for Saccharomyces cerevisiae phenylalanyltRNA synthetase, and Dr. James Cardelli and Floyd Galiano of the Feist-Weiller Cancer Center’s Innovative North Louisiana Experimental Therapeutics (INLET) program for assistance and use of the Synergy 4 Hybrid Microplate Reader. 1550

DOI: 10.1021/acs.biochem.5b01167 Biochemistry 2016, 55, 1541−1553

Article

Biochemistry

■

(15) Faulhammer, H. G., and Joshi, R. L. (1987) Structural features in aminoacyl-tRNAs required for recognition by elongation factor Tu. FEBS Lett. 217, 203−211. (16) Nissen, P., Kjeldgaard, M., Thirup, S., Clark, B. F., and Nyborg, J. (1996) The ternary complex of aminoacylated tRNA and EF-TuGTP. Recognition of a bond and a fold. Biochimie 78, 921−933. (17) Ellman, J. A., Mendel, D., and Schultz, P. G. (1992) Site-specific incorporation of novel backbone structures into proteins. Science (Washington, DC, U. S.) 255, 197−200. (18) Fujino, T., Goto, Y., Suga, H., and Murakami, H. (2013) Reevaluation of the D-amino acid compatibility with the elongation event in translation. J. Am. Chem. Soc. 135, 1830−1837. (19) Tan, Z., Forster, A. C., Blacklow, S. C., and Cornish, V. W. (2004) Amino acid backbone specificity of the Escherichia coli translation machinery. J. Am. Chem. Soc. 126, 12752−12753. (20) Dedkova, L. M., Fahmi, N. E., Golovine, S. Y., and Hecht, S. M. (2003) Enhanced D-amino acid incorporation into protein by modified ribosomes. J. Am. Chem. Soc. 125, 6616−6617. (21) Dedkova, L. M., Fahmi, N. E., Golovine, S. Y., and Hecht, S. M. (2006) Construction of modified ribosomes for incorporation of Damino acids into proteins. Biochemistry 45, 15541−15551. (22) Englander, M. T., Avins, J. L., Fleisher, R. C., Liu, B., Effraim, P. R., Wang, J., Schulten, K., Leyh, T. S., Gonzalez, R. L., Jr., and Cornish, V. W. (2015) The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center. Proc. Natl. Acad. Sci. U. S. A. 112, 6038−6043. (23) Yaremchuk, A., Kriklivyi, I., Tukalo, M., and Cusack, S. (2002) Class I tyrosyl-tRNA synthetase has a class II mode of cognate tRNA recognition. EMBO J. 21, 3829−3840. (24) Brick, P., Bhat, T. N., and Blow, D. M. (1989) Structure of tyrosyl-tRNA synthetase refined at 2.3 A resolution. Interaction of the enzyme with the tyrosyl adenylate intermediate. J. Mol. Biol. 208, 83− 98. (25) Brick, P., and Blow, D. M. (1987) Crystal structure of a deletion mutant of a tyrosyl-tRNA synthetase complexed with tyrosine. J. Mol. Biol. 194, 287−297. (26) Yang, X. L., Otero, F. J., Skene, R. J., McRee, D. E., Schimmel, P., and Ribas de Pouplana, L. (2003) Crystal structures that suggest late development of genetic code components for differentiating aromatic side chains. Proc. Natl. Acad. Sci. U. S. A. 100, 15376−15380. (27) Calendar, R., and Berg, P. (1966) The Catalytic Properties of Tyrosyl Ribonucleic Acid Synthetases from Escherichia coli and Bacillus subtilis. Biochemistry 5, 1690−1695. (28) Sheoran, A., and First, E. A. (2008) Activation of D-tyrosine by Bacillus stearothermophilus tyrosyl-tRNA synthetase: 2. Cooperative binding of ATP is limited to the initial turnover of the enzyme. J. Biol. Chem. 283, 12971−12980. (29) Sheoran, A., Sharma, G., and First, E. A. (2008) Activation of Dtyrosine by Bacillus stearothermophilus tyrosyl-tRNA synthetase: 1. Presteady-state kinetic analysis reveals the mechanistic basis for the recognition of D-tyrosine. J. Biol. Chem. 283, 12960−12970. (30) Oki, K., Sakamoto, K., Kobayashi, T., Sasaki, H. M., and Yokoyama, S. (2008) Transplantation of a tyrosine editing domain into a tyrosyl-tRNA synthetase variant enhances its specificity for a tyrosine analog. Proc. Natl. Acad. Sci. U. S. A. 105, 13298−13303. (31) First, E. A., and Fersht, A. R. (1993) Involvement of threonine 234 in catalysis of tyrosyl adenylate formation by tyrosyl-tRNA synthetase. Biochemistry 32, 13644−13650. (32) Sasaki, H. M., Sekine, S.-i., Sengoku, T., Fukunaga, R., Hattori, M., Utsunomiya, Y., Kuroishi, C., Kuramitsu, S., Shirouzu, M., and Yokoyama, S. (2006) Structural and mutational studies of the amino acid-editing domain from archaeal/eukaryal phenylalanyl-tRNA synthetase. Proc. Natl. Acad. Sci. U. S. A. 103, 14744−14749. (33) Ling, J., So, B. R., Yadavalli, S. S., Roy, H., Shoji, S., Fredrick, K., Musier-Forsyth, K., and Ibba, M. (2009) Resampling and editing of mischarged tRNA prior to translation elongation. Mol. Cell 33, 654− 660.

ABBREVIATIONS HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IPTG, isopropyl β-D-1-thiogalactopyranoside; Tris, Tris(hydroxymethyl)aminomethane; Tyr, tyrosine; NiNTA, nickel-nitrilotriacetic acid; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; CP1, connective polypeptide 1; FRSed, phenylalanyl-tRNA synthetase editing domain; TyrRS, tyrosyl-tRNA synthetase; TyrRS-WT, wild type tyrosyltRNA synthetase; TyrRS-FRSed, tyrosyl-tRNA synthetase variant containing the phenylalanyl-tRNA synthetase editing domain inserted into the CP1 domain; TyrRS·Tyr, tyrosyltRNA synthetase bound to tyrosine; TyrRS·ATP, tyrosyl-tRNA synthetase bound to ATP; TyrRS·Tyr·ATP, tyrosyl-tRNA synthetase bound to tyrosine and ATP; TyrRS·Tyr-AMP, tyrosyl-tRNA synthetase bound to the tyrosyl-adenylate intermediate; “·”, noncovalent interaction; “-”, covalent interaction

■

REFERENCES

(1) Jourdan, F., Lazzaroni, S., Mendez, B. L., Lo Cantore, P., de Julio, M., Amodeo, P., Iacobellis, N. S., Evidente, A., and Motta, A. (2003) A left-handed alpha-helix containing both L- and D-amino acids: the solution structure of the antimicrobial lipodepsipeptide tolaasin. Proteins: Struct., Funct., Genet. 52, 534−543. (2) Tancredi, T., Temussi, P. A., Picone, D., Amodeo, P., Tomatis, R., Salvadori, S., Marastoni, M., Santagada, V., and Balboni, G. (1991) New insights on mu/delta selectivity of opioid peptides: conformational analysis of deltorphin analogues. Biopolymers 31, 751−760. (3) Sela, M., and Zisman, E. (1997) Different roles of D-amino acids in immune phenomena. FASEB J. 11, 449−456. (4) Fujii, N., Momose, Y., Ishibashi, Y., Uemura, T., Takita, M., and Takehana, M. (1997) Specific racemization and isomerization of the aspartyl residue of alphaA-Crystallin due to UV-B irradiation. Exp. Eye Res. 65, 99−104. (5) Ingrosso, D., and Perna, A. F. (1998) D-amino acids in aging erythrocytes. EXS 85, 119−141. (6) Shapira, R., and Chou, C. H. (1987) Differential racemization of aspartate and serine in human myelin basic protein. Biochem. Biophys. Res. Commun. 146, 1342−1349. (7) Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C., and Schultz, P. G. (1989) A general method for site-specific incorporation of unnatural amino acids into proteins. Science (Washington, DC, U. S.) 244, 182−188. (8) Wang, L., Brock, A., Herberich, B., and Schultz, P. G. (2001) Expanding the genetic code of Escherichia coli. Science (Washington, DC, U. S.) 292, 498−500. (9) Calendar, R., and Berg, P. (1966) Purification and physical characterization of tyrosyl ribonucleic acid synthetases from Escherichia coli and Bacillus subtilis. Biochemistry 5, 1681−1690. (10) Soutourina, J., Plateau, P., and Blanquet, S. (2000) Metabolism of D-aminoacyl-tRNAs in Escherichia coli and Saccharomyces cerevisiae cells. J. Biol. Chem. 275, 32535−32542. (11) Fujimoto, K., Kubota, I., Yasuda-Kamatani, Y., Minakata, H., Nomoto, K., Yoshida, M., Harada, A., Muneoka, Y., and Kobayashi, M. (1991) Purification of achatin-I from the atria of the African giant snail, Achatina fulica, and its possible function. Biochem. Biophys. Res. Commun. 177, 847−853. (12) Passioura, T., and Suga, H. (2014) Reprogramming the genetic code in vitro. Trends Biochem. Sci. 39, 400−408. (13) Yamane, T., Miller, D. L., and Hopfield, J. J. (1981) Discrimination between D- and L-tyrosyl transfer ribonucleic acids in peptide chain elongation. Biochemistry 20, 7059−7064. (14) Eargle, J., Black, A. A., Sethi, A., Trabuco, L. G., and LutheySchulten, Z. (2008) Dynamics of Recognition between tRNA and elongation factor Tu. J. Mol. Biol. 377, 1382−1405. 1551

DOI: 10.1021/acs.biochem.5b01167 Biochemistry 2016, 55, 1541−1553

Article

Biochemistry

(55) Espenson, J. H. (1981) Chemical Kinetics and Reaction Mechanisms, 1st ed., pp 55−57, McGraw-Hill Book Company, New York. (56) Michaelis, L., and Menten, M. (1913) Die Kinetik der Invertinwirkung. Biochem. Z. 49, 333−369. (57) Michaelis, L., Menten, M. L., Johnson, K. A., and Goody, R. S. (2011) The original Michaelis constant: translation of the 1913 Michaelis-Menten paper. Biochemistry 50, 8264−8269. (58) Segal, I. H. (1976) Biochemical Calculations, John Wiley & Sons, Inc., New York. (59) Beckman Coulter, I. (1999) Isotope/DPM Libraries, In LS 6500 Scintillation System Operating Manual, pp 128−145, Beckman Coulter, Inc., Fullerton, CA. (60) Kotik-Kogan, O., Moor, N., Tworowski, D., and Safro, M. (2005) Structural basis for discrimination of L-phenylalanine from Ltyrosine by phenylalanyl-tRNA synthetase. Structure 13, 1799−1807. (61) Roy, H., Ling, J., Irnov, M., and Ibba, M. (2004) Post-transfer editing in vitro and in vivo by the beta subunit of phenylalanyl-tRNA synthetase. EMBO J. 23, 4639−4648. (62) Fersht, A. R., Ashford, J. S., Bruton, C. J., Jakes, R., Koch, G. L., and Hartley, B. S. (1975) Active site titration and aminoacyl adenylate binding stoichiometry of aminoacyl-tRNA synthetases. Biochemistry 14, 1−4. (63) Sheoran, A., and First, E. A. (2008) Activation of D-tyrosine by Bacillus stearothermophilus tyrosyl-tRNA synthetase: 2. Cooperative binding of ATP is limited to the initial turnover of the enzyme. J. Biol. Chem. 283, 12971−12980. (64) Fersht, A. R. (1975) Demonstration of two active sites on a monomeric aminoacyl-tRNA synthetase. Possible roles of negative cooperativity and half-of-the-sites reactivity in oligomeric enzymes. Biochemistry 14, 5−12. (65) Boniecki, M. T., and Martinis, S. A. (2012) Coordination of tRNA synthetase active sites for chemical fidelity. J. Biol. Chem. 287, 11285−11289. (66) Eldred, E. W., and Schimmel, P. R. (1972) Rapid deacylation by isoleucyl transfer ribonucleic acid synthetase of isoleucine-specific transfer ribonucleic acid aminoacylated with valine. J. Biol. Chem. 247, 2961−2964. (67) Fersht, A. R., and Dingwall, C. (1979) Evidence for the doublesieve editing mechanism in protein synthesis. Steric exclusion of isoleucine by valyl-tRNA synthetases. Biochemistry 18, 2627−2631. (68) Jakubowski, H., and Goldman, E. (1992) Editing of errors in selection of amino acids for protein synthesis. Microbiol. Rev. 56, 412− 429. (69) Schreier, A. A., and Schimmel, P. R. (1972) Transfer ribonucleic acid synthetase catalyzed deacylation of aminoacyl transfer ribonucleic acid in the absence of adenosine monophosphate and pyrophosphate. Biochemistry 11, 1582−1589. (70) Dulic, M., Cvetesic, N., Perona, J. J., and Gruic-Sovulj, I. (2010) Partitioning of tRNA-dependent editing between pre- and posttransfer pathways in class I aminoacyl-tRNA synthetases. J. Biol. Chem. 285, 23799−23809. (71) Perona, J. J., and Gruic-Sovulj, I. (2013) Synthetic and editing mechanisms of aminoacyl-tRNA synthetases. Top. Curr. Chem. 344, 1− 41. (72) Ling, J., Yadavalli, S. S., and Ibba, M. (2007) Phenylalanyl-tRNA synthetase editing defects result in efficient mistranslation of phenylalanine codons as tyrosine. RNA 13, 1881−1886. (73) Kiga, D., Sakamoto, K., Kodama, K., Kigawa, T., Matsuda, T., Yabuki, T., Shirouzu, M., Harada, Y., Nakayama, H., Takio, K., Hasegawa, Y., Endo, Y., Hirao, I., and Yokoyama, S. (2002) An engineered Escherichia coli tyrosyl-tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system. Proc. Natl. Acad. Sci. U. S. A. 99, 9715−9720. (74) Carta, R., and Tola, G. (1996) Solubilities of l-Cystine, lTyrosine, l-Leucine, and Glycine in Aqueous Solutions at Various pHs and NaCl Concentrations. J. Chem. Eng. Data 41, 414−417.

(34) First, E. A. (2015) A continuous spectrophotometric assay for monitoring adenosine 5′-monophosphate production. Anal. Biochem. 483, 34−39. (35) Richardson, C. J., and First, E. A. (2015) A continuous tyrosyltRNA synthetase assay that regenerates the tRNA substrate. Anal. Biochem. 486, 86−95. (36) Vetting, M. W., Hegde, S. S., and Blanchard, J. S. (2010) The structure and mechanism of the Mycobacterium tuberculosis cyclodityrosine synthetase. Nat. Chem. Biol. 6, 797−799. (37) Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D., and Bairoch, A. (2005) Protein Identification and Analysis Tools on the ExPASy Server, In The Proteomics Protocols Handbook, pp 571−605, Humana Press, New York. (38) Avis, J. M., Day, A. G., Garcia, G. A., and Fersht, A. R. (1993) Reaction of modified and unmodified tRNA(Tyr) substrates with tyrosyl-tRNA synthetase (Bacillus stearothermophilus). Biochemistry 32, 5312−5320. (39) Brown, R. S., Rubin, J. R., Rhodes, D., Guilley, H., Simoncsits, A., and Brownlee, G. G. (1978) The nucleoside sequence of tyrosine tRNA from Bacillus stearothermophilus. Nucleic Acids Res. 5, 23−36. (40) Xin, Y., Li, W., and First, E. A. (2000) The ’KMSKS’ motif in tyrosyl-tRNA synthetase participates in the initial binding of tRNA(Tyr). Biochemistry 39, 340−347. (41) Froelich, C. A., First, E. A. Dominant Intermediate CharcotMarie-Tooth disorder is not due to a catalytic defect in tyrosyl-tRNA synthetase, Biochemistry 201150, 7132−7145.10.1021/bi200989h (42) Cavaluzzi, M. J., Borer, P. N. (2004) Revised UV extinction coefficients for nucleoside-5′-monophosphates and unpaired DNA and RNA, Nucleic Acids Res. 32.13e10.1093/nar/gnh015 (43) Cantor, C. R., Warshaw, M. M., and Shapiro, H. (1970) Oligonucleotide interactions. 3. Circular dichroism studies of the conformation of deoxyoligonucleotides. Biopolymers 9, 1059−1077. (44) Wilkinson, A. J., Fersht, A. R., Blow, D. M., and Winter, G. (1983) Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation. Biochemistry 22, 3581−3586. (45) Fersht, A. R., Mulvey, R. S., and Koch, G. L. (1975) Ligand binding and enzymic catalysis coupled through subunits in tyrosyltRNA synthetase. Biochemistry 14, 13−18. (46) Wells, T. N., and Fersht, A. R. (1989) Protection of an unstable reaction intermediate examined with linear free energy relationships in tyrosyl-tRNA synthetase. Biochemistry 28, 9201−9209. (47) Sheoran, A., Sharma, G., and First, E. A. (2008) Activation of Dtyrosine by Bacillus stearothermophilus tyrosyl-tRNA synthetase: 1. Pre-steady-state kinetic analysis reveals the mechanistic basis for the recognition of D-tyrosine. J. Biol. Chem. 283, 12960−12970. (48) Ho, C. K., and Fersht, A. R. (1986) Internal thermodynamics of position 51 mutants and natural variants of tyrosyl-tRNA synthetase. Biochemistry 25, 1891−1897. (49) Hofstee, B. H. (1959) Non-inverted versus inverted plots in enzyme kinetics. Nature 184, 1296−1298. (50) Hill, A. V. (1910) The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J. Physiol. 40, iv− vii. (51) Fersht, A. R., and Jakes, R. (1975) Demonstration of two reaction pathways for the aminoacylation of tRNA. Application of the pulsed quenched flow technique. Biochemistry 14, 3350−3356. (52) Scatchard, G. (1942) Equilibrium Thermodynamics and Biological Chemistry. Science (Washington, DC, U. S.) 95, 27−32. (53) Fersht, A. R., Knill-Jones, J. W., Bedouelle, H., and Winter, G. (1988) Reconstruction by site-directed mutagenesis of the transition state for the activation of tyrosine by the tyrosyl-tRNA synthetase: a mobile loop envelopes the transition state in an induced-fit mechanism. Biochemistry 27, 1581−1587. (54) Wells, T. N., Ho, C. K., and Fersht, A. R. (1986) Free energy of hydrolysis of tyrosyl adenylate and its binding to wild-type and engineered mutant tyrosyl-tRNA synthetases. Biochemistry 25, 6603− 6608. 1552

DOI: 10.1021/acs.biochem.5b01167 Biochemistry 2016, 55, 1541−1553

Article

Biochemistry (75) Sasaki, H. M., Sekine, S., Sengoku, T., Fukunaga, R., Hattori, M., Utsunomiya, Y., Kuroishi, C., Kuramitsu, S., Shirouzu, M., and Yokoyama, S. (2006) Structural and mutational studies of the amino acid-editing domain from archaeal/eukaryal phenylalanyl-tRNA synthetase. Proc. Natl. Acad. Sci. U. S. A. 103, 14744−14749. (76) Simonson, T., Ye-Lehmann, S., Palmai, Z., Amara, N., WydauDematteis, S., Bigan, E., Druart, K., Moch, C., and Plateau, P. (2016) Redesigning the stereospecificity of tyrosyl-tRNA synthetase. Proteins: Struct., Funct., Genet. 84, 240−253.

1553

DOI: 10.1021/acs.biochem.5b01167 Biochemistry 2016, 55, 1541−1553