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• W2040897483 abstract "Lysyl-tRNA synthetase from Bacillus stearothermophilus (B.s. LysRS) (EC 6.1.1.6) catalyzes aminoacylation of tRNALys withl-lysine, in which l-lysine was first activated with ATP to yield an enzyme (lysyladenylate complex), and then the lysine molecule was transferred from the complex to tRNALys. B.s. LysRS is a homodimeric enzyme with a subunit that consists of two domains, an N-terminal tRNA anticodon-binding domain (TAB-ND: Ser1-Pro144) and a C-terminal Class II-specific catalytic domain (CAT-CD: Lys151-Lys493). CAT-CD alone retained catalytic activity, although at a low level; TAB-ND alone showed no activity. Size exclusion chromatography revealed that CAT-CD exists as a dimer, whereas TAB-ND was a monomer. The formation of a complex consisting of these domains was detected with the guidance of surface plasmon resonance. In accordance with this, the addition of TAB-ND to CAT-CD significantly enhanced both the l-lysine activation and the tRNA aminoacylation reactions. Kinetic analysis showed that deletion of TAB-ND resulted in a significant destabilization of the transition state of CAT-CD in the l-lysine activation reaction but had little effect on the ground state of substrate binding. A significant role of a cross-subunit interaction in the enzyme between TAB-ND and CAT-CD was proposed for the stabilization of the transition state in the l-lysine activation reaction. Lysyl-tRNA synthetase from Bacillus stearothermophilus (B.s. LysRS) (EC 6.1.1.6) catalyzes aminoacylation of tRNALys withl-lysine, in which l-lysine was first activated with ATP to yield an enzyme (lysyladenylate complex), and then the lysine molecule was transferred from the complex to tRNALys. B.s. LysRS is a homodimeric enzyme with a subunit that consists of two domains, an N-terminal tRNA anticodon-binding domain (TAB-ND: Ser1-Pro144) and a C-terminal Class II-specific catalytic domain (CAT-CD: Lys151-Lys493). CAT-CD alone retained catalytic activity, although at a low level; TAB-ND alone showed no activity. Size exclusion chromatography revealed that CAT-CD exists as a dimer, whereas TAB-ND was a monomer. The formation of a complex consisting of these domains was detected with the guidance of surface plasmon resonance. In accordance with this, the addition of TAB-ND to CAT-CD significantly enhanced both the l-lysine activation and the tRNA aminoacylation reactions. Kinetic analysis showed that deletion of TAB-ND resulted in a significant destabilization of the transition state of CAT-CD in the l-lysine activation reaction but had little effect on the ground state of substrate binding. A significant role of a cross-subunit interaction in the enzyme between TAB-ND and CAT-CD was proposed for the stabilization of the transition state in the l-lysine activation reaction. aminoacyl-tRNA synthetase lysyl-tRNA synthetase fromBacillus stearothermophilus C-terminal catalytic domain of B.s. LysRS molar absorption coefficient at 280 nm binding standard free energy activation free energy association rate constant dissociation rate constant Michaelis constant for X N-terminal tRNA anticodon-binding domain of B.s. LysRS resonance unit the excitation wavelength surface plasmon resonance; other aminoacyl-tRNA synthetases were also abbreviated with the three-letter abbreviation of their specific amino acid followed by RS AaRS1 catalyzes the ligation of an amino acid to the cognate tRNA, generally according to the following (Reaction Scheme 1), E+AA+ATP⇔E·AA∼AMP+PPiE·AA∼AMP+tRNA→E+AMP+AA­tRNAReaction Scheme 1 where AA denotes the amino acid; E, aaRS; PPi, inorganic pyrophosphate; and E·AA∼AMP, an aaRS·aminoacyladenylate complex. Because the tRNA aminoacylation reaction is critical for the fidelity of translation of genetic information into the structure of a protein, aaRSs must have gained a high degree of substrate specificity for each heterogeneous substrate during evolution. Recent progress in x-ray crystallographic analysis has revealed that aaRSs can be classified into two groups according to their active site topology. Class I aaRSs possess a catalytic core composed of a Rossmann fold, whereas Class II aaRSs possess a catalytic core consisting of antiparallel β-sheets (1Fersht A.R. Kaethner M.M. Biochemistry. 1976; 15: 818-823Crossref PubMed Scopus (83) Google Scholar, 2Moras D. Trends Biochem. Sci. 1992; 17: 159-164Abstract Full Text PDF PubMed Scopus (199) Google Scholar). AaRSs are considered to have emerged at an early stage in the development of the contemporary system of protein synthesis (3Schimmel P. Giegé R. Moras D. Yokoyama S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8763-8768Crossref PubMed Scopus (347) Google Scholar). In addition, it was reported that several isolated catalytic domains of aaRS retained full or part of the amino acid activation activity (4Cassio D. Waller J.P. Eur. J. Biochem. 1971; 20: 283-300Crossref PubMed Scopus (186) Google Scholar, 5Jasin M. Regan L. Schimmel P. Nature. 1983; 306: 441-447Crossref PubMed Scopus (117) Google Scholar, 6Borel F. Vincent C. Leberman R. Härtlein M. Nucleic Acids Res. 1994; 22: 2963-2969Crossref PubMed Scopus (47) Google Scholar, 7Augustine J. Francklyn C. Biochemistry. 1997; 36: 3473-3482Crossref PubMed Scopus (32) Google Scholar, 8Sankaranarayanan R. Dock-Bregeon A-C. Romby P. Caillet J. Springer M. Rees B. Ehresmann C. Ehresmann B. Moras D. Cell. 1999; 97: 371-381Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Together, these results have led to the proposal that the modern aaRS evolved from a primordial ancestor that had either of the two types of the class-defining domains, by getting the idiosyncratic region of each aaRS (3Schimmel P. Giegé R. Moras D. Yokoyama S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8763-8768Crossref PubMed Scopus (347) Google Scholar, 9Rould M.A. Perona J.J. Söll D. Steiz T.A. Science. 1989; 246: 1135-1142Crossref PubMed Scopus (803) Google Scholar, 10Steer B.A. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13644-13649Crossref PubMed Scopus (15) Google Scholar). In fact, the typical insertion in Class I aaRSs of the variable connective polypeptides (11Hou Y.-M. Shiba K. Mottes C. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 976-980Crossref PubMed Scopus (91) Google Scholar) is responsible for the stabilization of the transition state of methionine activation as well as methionine transfer to the 3′ end of tRNA in Escherichia coli MetRS (12Fourmy D. Mechulam Y. Blanquet S. Biochemistry. 1995; 34: 15681-15688Crossref PubMed Scopus (22) Google Scholar), and also for the hydrolysis of misacylated Val-tRNAIle in Thermus thermophilus IleRS (13Nureki O. Vassylyev D.G. Teteno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Science. 1998; 280: 578-581Crossref PubMed Scopus (315) Google Scholar). On the other hand, the crystallographic structures of the aaRS·tRNA complex (8Sankaranarayanan R. Dock-Bregeon A-C. Romby P. Caillet J. Springer M. Rees B. Ehresmann C. Ehresmann B. Moras D. Cell. 1999; 97: 371-381Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 9Rould M.A. Perona J.J. Söll D. Steiz T.A. Science. 1989; 246: 1135-1142Crossref PubMed Scopus (803) Google Scholar, 14Ruff M. Krishnaswamy S. Boeglin M. Poterszman A. Mitschler A. Podjarny A. Rees B. Thierry J.C. Moras D. Science. 1991; 252: 1682-1689Crossref PubMed Scopus (593) Google Scholar, 15Cusack S. Yaremchuk A. Tukalo M. EMBO J. 1996; 15: 6321-6334Crossref PubMed Scopus (150) Google Scholar) have revealed that an aaRS prepares a non-catalytic domain for the interaction with the anticodon stem/loop of the cognate tRNA and that each anticodon-binding domain shows considerable variation in its structure. Thus far, SerRS has been an exceptional case in which the anticodon stem/loop of the tRNA was not recognized by aaRS. In this system, the N-terminal non-catalytic domain interacts with an abnormally long variable arm of tRNASer(16Sampson J.R. Saks M.E. Nucleic Acids Res. 1993; 21: 4467-4475Crossref PubMed Scopus (77) Google Scholar). Recently, it has been suggested that the interface between the anticodon-binding domain and the catalytic domain plays an important role in the tRNA-dependent conformational change in the active site, not only in GlnRS, which requires the cognate tRNA for the amino acid activation reaction (17Sherman J.M. Thomann H.-U. Söll D. J. Mol. Biol. 1996; 256: 818-828Crossref PubMed Scopus (29) Google Scholar), but also in MetRS, which does not require it (3Schimmel P. Giegé R. Moras D. Yokoyama S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8763-8768Crossref PubMed Scopus (347) Google Scholar, 18Alexader R.W. Schimmel P. Biochemistry. 1999; 38: 16359-16365Crossref PubMed Scopus (20) Google Scholar). Furthermore, it has been reported that an accurate anticodon-aaRS interaction was essential for enhancement of the catalytic constant of aminoacylation (10Steer B.A. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13644-13649Crossref PubMed Scopus (15) Google Scholar, 19Jahn M. Rogers M.J. Söll D. Nature. 1991; 352: 258-260Crossref PubMed Scopus (169) Google Scholar, 20Pütz J. Puglisi J.D. Florentz C. Giegé R. EMBO J. 1993; 12: 2949-2957Crossref PubMed Scopus (57) Google Scholar, 21Eriani G. Gangloff J. J. Mol. Biol. 1999; 291: 761-773Crossref PubMed Scopus (31) Google Scholar). These results suggest that in some aaRSs, the signal generated by the appropriate interaction between the anticodon of tRNA and the anticodon-binding domain was transmitted to the active site through either the aaRS or the tRNA molecule (10Steer B.A. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13644-13649Crossref PubMed Scopus (15) Google Scholar, 17Sherman J.M. Thomann H.-U. Söll D. J. Mol. Biol. 1996; 256: 818-828Crossref PubMed Scopus (29) Google Scholar, 18Alexader R.W. Schimmel P. Biochemistry. 1999; 38: 16359-16365Crossref PubMed Scopus (20) Google Scholar). In contrast to the tRNA-dependent domain-domain interaction, the role of the interaction between the anticodon-binding domain and the catalytic domain has never been reported in the absence of tRNA. Thus far, only for dimeric HisRS (Class IIa), both the catalytic domain and the non-catalytic domain (this domain has not yet been proven to be the anticodon-binding domain) have been purified separately (7Augustine J. Francklyn C. Biochemistry. 1997; 36: 3473-3482Crossref PubMed Scopus (32) Google Scholar). In this system, it was shown that in both the tRNA-independent amino acid activation and the aminoacylation reactions, the C-terminal non-catalytic domain contributed significantly to the stabilization of the transition state, but not to the ground state of substrate binding. In this case, however, because the catalytic domain of HisRS was purified as monomer, it was not clear whether the observed destabilization of the transition state upon deletion of the non-catalytic domain was because of the dissociation of the catalytic domain into a monomer or because of the loss of the interaction between the catalytic domain and the non-catalytic domain. In the course of the studies on B.s. LysRS (l-lysine:tRNALys ligase (AMP forming); EC6.1.1.6), a Class II enzyme, from Bacillus stearothermophilus (22Takita T. Ohkubo Y. Shima H. Muto T. Shimizu N. Sukata T. Ito H. Saito Y. Inouye K. Hiromi K. Tonomura B. J. Biochem. (Tokyo). 1996; 119 (Correction): 680-689Crossref PubMed Scopus (14) Google ScholarJ. Biochem. (Tokyo). 1998; 123: 1218Crossref Google Scholar, 23Takita T. Hashimoto S. Ohkubo Y. Muto T. Shimizu N. Sukata T. Inouye K. Hiromi K. Tonomura B. J. Biochem. (Tokyo). 1997; 121: 244-250Crossref PubMed Scopus (7) Google Scholar, 24Takita T. Akita E. Inouye K. Tonomura B. J. Biochem. (Tokyo). 1998; 124: 45-50Crossref PubMed Scopus (9) Google Scholar, 25Takita T. Shimizu N. Sukata T. Hashimoto S. Akita E. Yokota Y. Esaki N. Soda K. Inouye K. Tonomura B. Biosci. Biotechnol. Biochem. 2000; 64: 432-437Crossref PubMed Scopus (2) Google Scholar), we have dissected the enzyme into N-terminal and C-terminal domains. By analogy with the crystallographic structure of Escherichia coli LysRS(U) (26Onesti S. Miller A.D. Brick P. Structure. 1995; 3: 163-176Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), homodimericB.s. LysRS (molecular mass of the subunit is 57,273 Da (25Takita T. Shimizu N. Sukata T. Hashimoto S. Akita E. Yokota Y. Esaki N. Soda K. Inouye K. Tonomura B. Biosci. Biotechnol. Biochem. 2000; 64: 432-437Crossref PubMed Scopus (2) Google Scholar)) was considered to have a simple modular organization in which the C-terminal catalytic domain (CAT-CD) was joined through a short flexible linker to the N-terminal anticodon-binding domain (TAB-ND) (Fig. 1). In this study, we have succeeded in purifying CAT-CD as a dimer. The aim of this study was to elucidate the role of the interaction between the catalytic domain and the anticodon-binding domain in the tRNA-independent l-lysine activation reaction, and to elucidate the mechanism of the transition state stabilization in the enzymatic reaction. The recombinant form of wild-type B.sLysRS was purified from E. coli BL21(DE3) cells by the method reported previously (25Takita T. Shimizu N. Sukata T. Hashimoto S. Akita E. Yokota Y. Esaki N. Soda K. Inouye K. Tonomura B. Biosci. Biotechnol. Biochem. 2000; 64: 432-437Crossref PubMed Scopus (2) Google Scholar). The enzyme concentration was determined spectrophotometrically with ε280 of 70,600m−1 cm−1 at pH 8.0, 25° C (25Takita T. Shimizu N. Sukata T. Hashimoto S. Akita E. Yokota Y. Esaki N. Soda K. Inouye K. Tonomura B. Biosci. Biotechnol. Biochem. 2000; 64: 432-437Crossref PubMed Scopus (2) Google Scholar). Streptomyces subtilisin inhibitor was a gift from Dr. B. Tonomura. Superoxide dismutase from bovine erythrocytes was the product of Wako Pure Chemical. Gel filtration molecular weight markers were purchased from Sigma. l-[4,5-3H]Lysine and [32P]pyrophosphate were the products of AmershamBiosciences; ATP (disodium salt) was from Sigma; glass microfiber filter (GF/C) was from Whatman Ltd.; Q-Sepharose HP was fromAmersham Biosciences; and Gigapite was from Toagosei. DEAE-Toyopearl 650M, TSK gel G3000 SWXL prepacked column (inner diameter 7.8 mm × 30 cm), and TSK gel phenyl-5PW (inner diameter 7.5 mm × 7.5 cm) were purchased from Tosoh. BIAcore chemical activation reagents were obtained from Amersham Biosciences. Sensor chips (BIAcore CM5 Research grade) and HBS buffer (10 mm HEPES buffer (pH 7.4) containing 0.15 mNaCl, 3 mm EDTA, and 0.005% Tween 20) were the products of BIAcore. All other chemicals were of reagent grade. Based on the amino acid sequence alignment between E. coli LysRS(U) and B.s. LysRS (25Takita T. Shimizu N. Sukata T. Hashimoto S. Akita E. Yokota Y. Esaki N. Soda K. Inouye K. Tonomura B. Biosci. Biotechnol. Biochem. 2000; 64: 432-437Crossref PubMed Scopus (2) Google Scholar), the anticodon-binding domain (Ser1-Pro144) and the catalytic domain (Lys151-Lys493) of B.s. LysRS were designed to be expressed in the form with an N-terminal T7 Tag sequence as TAB-ND and CAT-CD, respectively (Table I). Two sense N-terminal primers with BamHI restriction sites, 5′-TACCACGGATCCAAAGATATCGAGCAG-3′ (N1-primer) and 5′-AGGTGGATCCGGTATGAGCCATG-3′ (N2-primer), and two antisense C-terminal primers with BamHI restriction sites, 5′CGCAACAGGGGAGGATCCTGGTTA-3′ (C1-primer) and 5′-CGTGGGATCCTTACGGCAGCGGA-3′ (C2-primer), were synthesized. By PCR reaction with pBLX45 containing the B.s.LysRS gene (25Takita T. Shimizu N. Sukata T. Hashimoto S. Akita E. Yokota Y. Esaki N. Soda K. Inouye K. Tonomura B. Biosci. Biotechnol. Biochem. 2000; 64: 432-437Crossref PubMed Scopus (2) Google Scholar) and Pfu DNA polymerase, the genes corresponding to TAB-ND and CAT-CD were amplified with N2- and C2-primers and N1- and C1-primers, respectively. The amplified DNA fragments were digested with BamHI and inserted into the corresponding site of the pET11a expression vector. After transformation of E. coli BL21(DE3) cells, the DNA sequences of the TAB-ND and CAT-CD genes were determined with an ABI PRISM 377 Sequencer (Applied Biosystems).Table IStructural information for B.s. LysRS, TAB-ND, and CAT-CDN-terminal T7 tag sequenceAmino acid residuesQuaternary structureMrNumber ofTrpTyrB.s.LysRSSer1–Lys493Dimer57,273 × 22 × 217 × 2TAB-NDASMTGGQQMGRGSGMSer1–Pro144Monomer17,96504CAT-CDASMTGGQQMGRGSLys151–Lys493Dimer41,286 × 22 × 212 × 2 Open table in a new tab TAB-ND and CAT-CD were induced at 37 °C with 1 mmisopropyl-1-thio-β-d-galactoside in E. coliBL21(DE3) cells grown in Luria-Bertani medium containing 100 μg/ml ampicillin. TAB-ND and CAT-CD were purified in the same manner as the intact enzyme until the ammonium sulfate precipitation (60% saturation), except for omitting the heat treatment at 55 °C (25Takita T. Shimizu N. Sukata T. Hashimoto S. Akita E. Yokota Y. Esaki N. Soda K. Inouye K. Tonomura B. Biosci. Biotechnol. Biochem. 2000; 64: 432-437Crossref PubMed Scopus (2) Google Scholar). The solution was loaded to a DEAE-Toyopearl 650M column and eluted with a linear gradient of 0–1 m NaCl. The CAT-CD fraction was loaded to a Gigapite column and eluted with a linear gradient of 10–400 mm phosphate buffer (pH 6.8). On the other hand, the TAB-ND fraction was loaded to a Red Toyopearl column and eluted with a linear gradient of 0–1.0 m NaCl. Each dialyzed fraction was loaded to a Q-Sepharose HP column and eluted with a linear gradient of 0–1.0 m NaCl. After dialysis against 100 mm Tris-HCl (pH 8.0), each fraction was applied to a TSK gel phenyl-5PW column in the high performance liquid chromatography system, and proteins were eluted with a linear gradient of ammonium sulfate from 1.2 to 0 m. The TAB-ND and CAT-CD fractions were further applied to a TSK gel G3000 SWXL column equipped with TSK guard column SWXL. All operations were done at 4 °C except the high performance liquid chromatography operations that were carried out at room temperature. Protein concentration was measured by the method of Lowry et al. (27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar, 28Shrake A. Rupley J.A. J. Mol. Biol. 1973; 79: 351-371Crossref PubMed Scopus (1000) Google Scholar) with crystalline bovine serum albumin as the standard. At pH 8.0 and 25 °C, the estimated ε280 values of the TAB-ND monomer (17,965 Da) and CAT-CD dimer (82,571 Da) were 5,200 and 45,000 m−1cm−1, respectively. The concentrations of the TAB-ND monomer and CAT-CD dimer were determined spectrophotometrically using these values. The amino acid sequences of the N termini of TAB-ND and CAT-CD were determined using a gas-phase protein sequencer (Applied Biosystems model 477A) with an on-line PTH analyzer (Applied Biosystems model 120A). 200 ng of purified TAB-ND and CAT-CD were analyzed by SDS-PAGE on 10–20% gradient gels at a constant current of 40 mA for 90 min, and visualized by Coomassie staining. The quaternary structures of TAB-ND and CAT-CD were investigated by size exclusion chromatography on a TSK gel G3000 SWXL column with a flow-rate of 0.8 ml/min. The running buffer was 100 mm Tris-HCl buffer (pH 8.0) containing 10 mm MgCl2. All measurements were done in 100 mm Tris-HCl buffer (pH 8.0) containing 10 mm MgCl2. UV absorption spectra and far-UV CD spectra were measured at 25 °C with a Shimadzu spectrophotometer UV-2200 and a Shimadzu-Aviv circular dichroism spectrophotometer model 202, respectively. Fluorescence spectra were measured at 30 °C with a Hitachi fluorescence spectrophotometer 850. Real-time interaction between TAB-ND and CAT-CD was detected with the guidance of surface plasmon resonance (SPR) at 25 °C using a BIAcore 2000. Activation of the carboxylmethylated dextran in the sensor chip was carried out by mixing equal volumes of 100 mm N-hydroxysuccinimide in water and 400 mm N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride in water, and injecting the mixture at 20 μl/min for 7 min. 50 μg/ml TAB-ND and 2,000 μg/ml CAT-CD dissolved in 10 mm acetic acid buffer (pH 5.0) were separately injected over the activated surface of the sensor chip for 7 min at a flow rate of 20 μl/min. The unreacted sites of the sensor chip were masked by the injection for 7 min of 1 m ethanolamine hydrochloride adjusted to pH 8.5 using NaOH. The molecules that non-specifically bound to the sensor chip surface were removed by washing with HBS buffer until the value of RU became nearly constant. The signals obtained were ∼1,000 RU, which were in the permitted range for determining kinetic constants. In the present analysis, no regeneration procedure was applied because of a significant loss in the binding capacity of each domain of B.s. LysRS. Binding experiments were carried out by injecting CAT-CD in HBS buffer at 40 μl/min onto the sensor surface on which TAB-ND was immobilized, and vice versa. k a, k d, and K dwere estimated by assuming a simple bimolecular binding equilibrium (see Supplemental Materials). In the apparent association-reaction, the formation of the complex was represented as follows. R=ka[B]0Rmaxka[B]0+kd·(1−e−(ka[B]0+kd)·t)Equation 1 When R max is defined in RU as the maximum binding capacity of one domain (A) immobilized, which was determined by saturation with the other free domain (B), [AB] corresponds toR , which was the amount of bound domain B shown in RU at time t. The values ofk a[B]0 +k d andk a[B]0 R max were estimated by non-linear fitting of Equation 1. A linear plot ofk a[B]0 +k d versus [B]0yielded a slope of k a and an intercept ofk d. On the other hand, in the apparent dissociation reaction, the disappearance of the complex was represented as follows, lnR0Rn=kd(tn−t0)Equation 2 where R n was R at timet n, and R 0 is R at an arbitrary starting time, t 0. Thek d value was obtained as the linear slope of theln(R 0/R n)versus (t n-t 0) plot. The l-lysine activation reaction was measured by using thel-lysine-dependent ATP-PPi exchange reaction at pH 8.0, 37 °C, as reported previously (22Takita T. Ohkubo Y. Shima H. Muto T. Shimizu N. Sukata T. Ito H. Saito Y. Inouye K. Hiromi K. Tonomura B. J. Biochem. (Tokyo). 1996; 119 (Correction): 680-689Crossref PubMed Scopus (14) Google ScholarJ. Biochem. (Tokyo). 1998; 123: 1218Crossref Google Scholar). The standard reaction mixture contained in 0.5 ml: 100 mm Tris-HCl buffer (pH 8.0), 10 mm MgCl2, ATP, l-lysine, and PPi (9.1 mCi/mmol). Kinetic parameters were calculated by the nonlinear least-squares method with KaleidaGraph (Synergy software).K m,Lys was estimated over a range ofl-lysine concentrations from 10 to 200 μm, whereas K m,ATP was estimated over a range of ATP concentrations from 20 to 500 μm.K m,PPi was estimated over a range of PPi concentrations from 30 to 500 μm. In all cases, the initial concentration of the other two substrates was 1 mm. 1 mm might not be the saturating concentration for ATP (see Table II), but this concentration was chosen after considering the ratio of ATP to Mg2+. Enzyme concentrations were 5 nm B.s. LysRS, 500 nm CAT-CD, and 75 nm TAB-ND plus 5 nm CAT-CD.Table IIKinetic and static parameters of B.s. LysRS, CAT-CD, and the TAB-ND·CAT-CD complexATP-PPi exchange reactionk catK m,LysK m,ATPK m,PPi(s−1)2-ak cat values were determined in the presence of 1 mm l-lysine plus 1 mm PPi.(μm)B.s.LysRS46.4 ± 1.02-b[E]0 = 5 nm.14.1 ± 1.8227 ± 1050.2 ± 5.3TAB-NDND2-cND, not determined.CAT-CD0.098 ± 0.0162-d500 nm.57.2 ± 19.3462 ± 12618.3 ± 3.0TAB-ND + CAT-CD17.1 ± 0.62-e75 nm TAB-ND + 5 nm CAT-CD.13.2 ± 2.5184 ± 1335.7 ± 5.314.7 ± 0.72-fEstimated in Fig. 4.Aminoacylation reactionk catK m,LysK m,ATP(s −1)2-gk cat values were determined in the presence of 1 mm l-lysine plus 20 A 260units of tRNA.(μm)B.s.LysRS3.48 ± 0.262-h[E]0 = 0.5 nm.20.9 ± 4.560.8 ± 4.8TAB-NDNDCAT-CD0.00012 ± 0.000022-i300 nm.60.5 ± 21.390.2 ± 24.5TAB-ND + CAT-CD0.047 ± 0.0022-j150 nM TAB-ND + 10 nm CAT-CD.12.7 ± 4.152.0 ± 8.40.041 ± 0.0052-fEstimated in Fig. 4.K d of TAB-ND and CAT-CD(nm)SPR16.4 ± 1.22-kEstimated in Fig. 3.ATP-PPi exchange reaction30.7 ± 4.02-fEstimated in Fig. 4.Aminoacylation reaction41.8 ± 15.22-fEstimated in Fig. 4.Each enzyme activity was measured at pH 8.0 and 37°C in 100 mM Tris-HCl buffer containing 10 mmMgCl2. k cat values were determined with the ATP concentration varied.2-a k cat values were determined in the presence of 1 mm l-lysine plus 1 mm PPi.2-b [E]0 = 5 nm.2-c ND, not determined.2-d 500 nm.2-e 75 nm TAB-ND + 5 nm CAT-CD.2-f Estimated in Fig. 4.2-g k cat values were determined in the presence of 1 mm l-lysine plus 20 A 260units of tRNA.2-h [E]0 = 0.5 nm.2-i 300 nm.2-j 150 nM TAB-ND + 10 nm CAT-CD.2-k Estimated in Fig. 3. Open table in a new tab Each enzyme activity was measured at pH 8.0 and 37°C in 100 mM Tris-HCl buffer containing 10 mmMgCl2. k cat values were determined with the ATP concentration varied. The tRNA aminoacylation reaction was measured at pH 8.0, 37 °C, as reported previously (22Takita T. Ohkubo Y. Shima H. Muto T. Shimizu N. Sukata T. Ito H. Saito Y. Inouye K. Hiromi K. Tonomura B. J. Biochem. (Tokyo). 1996; 119 (Correction): 680-689Crossref PubMed Scopus (14) Google ScholarJ. Biochem. (Tokyo). 1998; 123: 1218Crossref Google Scholar). The standard reaction mixture contained in 0.5 ml: 100 mmTris-HCl buffer (pH 8.0), 10 mm MgCl2, ATP,l-[3H]lysine (40 mCi/mmol), and 20A 260 units of E. coli tRNA.K m,Lys was estimated over a range ofl-lysine concentrations from 5 to 100 μm in the presence of 1 mm ATP and 20 A 260units of tRNA, whereas K m,ATP was estimated over a range of ATP concentrations from 50 to 300 μm in the presence of 1 mml-lysine and 20 A 260 units of tRNA. Enzyme concentrations were 0.5 nm B.s. LysRS, 300 nm CAT-CD, and 150 nm TAB-ND plus 10 nm CAT-CD. The radioactivity in both assays was measured in an Aloka Liquid Scintillation Counter LSC-5100. B.s LysRS consists of 493 amino acid residues and the amino acid sequence has relatively high homology (53%) toE. coli LysRS(U) (504 residues) (25Takita T. Shimizu N. Sukata T. Hashimoto S. Akita E. Yokota Y. Esaki N. Soda K. Inouye K. Tonomura B. Biosci. Biotechnol. Biochem. 2000; 64: 432-437Crossref PubMed Scopus (2) Google Scholar). Therefore, to obtain information on the domain-domain interactions of B.s. LysRS, each contact area between the domains of E. coli LysRS(U) was estimated based on the 2–5 Å resolution structure of the complex with the substrate l-lysine (26Onesti S. Miller A.D. Brick P. Structure. 1995; 3: 163-176Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) (Fig.1). A dimer of E. coliLysRS(U) was chosen, and hydrogens were added to the groups ionized at pH 8. After removing the l-lysine substrate and water molecules, the accessible surface area of the free dimer enzyme was estimated with Insight II/Homology (Biosym Technologies, San Diego, CA) by rolling a sphere of 1–4 Å radius corresponding to a water molecule around the enzyme (27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). In the same way, the accessible surface areas of NA, NB, CA, CB, (NA·CA), (NB·CB), (NA·CB), (NB·CA), and (NA·CA·NB·CB) were estimated, where the N-terminal domain (Phe14-Pro153) and C-terminal domain (Asp161-Pro502) in subunit A of the E. coli LysRS(U) dimer were denoted as NA and CA, respectively, and in subunit B as NB and CB. Both TAB-ND and CAT-CD were more highly expressed in E. coli BL21(DE3) cells compared with the intact enzyme, and were purified to homogeneity as judged by SDS-PAGE (Fig. 2). Their migrations on SDS-PAGE were consistent with the molecular mass of TAB-ND, 17,965 Da, and that of CAT-CD, 41,286 Da (TableI). N-terminal amino acid sequences of TAB-ND and CAT-CD were determined as ASMTGGQQMGRGSGMSHEELand ASMTGGQQMGRGSKDIEQRY, respectively, in complete accordance with those expected from their DNA sequences (Table I). The underlined regions show the sequence from Ser1 to Leu5 of TAB-ND and from Lys151 to Tyr157 of CAT-CD. Size exclusion chromatography (Supplemental Materials) indicated that TAB-ND existed as a monomer but CAT-CD was a dimer under the conditions used, because the molecular masses of TAB-ND and CAT-CD were estimated to be 13,500 and 88,300 Da, respectively (Supplemental Materials). UV absorption spectra of TAB-ND, CAT-CD, and the intact B.s.LysRS are shown in the Supplemental Materials. The spectra of TAB-ND have a shoulder at 283 nm in analogy to tyrosine spectra. Estimated ε280 values at pH 8.0, 25 °C, 5,200m−1 cm−1 for the TAB-ND monomer and 45,000 m−1 cm−1 for the CAT-CD dimer were roughly consistent with 4,793 and 50,988m−1 cm−1, respectively, which were calculated by the numbers and ε280 values of the Phe, Tyr, and Trp residues. Fluorescence emission spectra of TAB-ND, CAT-CD, and the intact B.s. LysRS are shown in the Supplemental Materials. The emission spectra of TAB-ND at an excitation wavelength (λex) of 280 nm has a λmax value of 304 nm, corresponding to 306 nm ofN-acetyl-l-tyrosine ethyl ester, whereas no appreciable fluorescence was observed at λex = 295 nm. These data were in accordance with the fact that TAB-ND contains Tyr residues but no Trp residues, as deduced from the primary structure ofB.s. LysRS (Table I). On the other hand, the emission spectra of CAT-CD at λex = 295 nm indicates the presence of Trp residues and that the λmax value of 336 nm was very close to the corresponding 335 nm of the intact B.s.LysRS (26Onesti S. Miller A.D. Brick P. Structure. 1995; 3: 163-176Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). CD spectra of TAB-ND, CAT-CD, TAB-ND plus CAT-CD, and B.s LysRS, as well as the synthesized spectra of TAB-ND plus CAT-CD are shown in the Supplemental Materials. Because the sum of the number of amin" @default.
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• W2040897483 title "Transition State Stabilization by the N-terminal Anticodon-binding Domain of Lysyl-tRNA Synthetase" @default.
• W2040897483 cites W110138870 @default.
• W2040897483 cites W1555552538 @default.
• W2040897483 cites W1775749144 @default.
• W2040897483 cites W1972356897 @default.
• W2040897483 cites W1973453628 @default.
• W2040897483 cites W1983263986 @default.
• W2040897483 cites W1986719399 @default.
• W2040897483 cites W1987589325 @default.
• W2040897483 cites W1988283573 @default.
• W2040897483 cites W1992932889 @default.
• W2040897483 cites W1993855691 @default.
• W2040897483 cites W1994056136 @default.
• W2040897483 cites W1995616206 @default.
• W2040897483 cites W2001686780 @default.
• W2040897483 cites W2001900040 @default.
• W2040897483 cites W2004416250 @default.
• W2040897483 cites W2004748578 @default.
• W2040897483 cites W2006755511 @default.
• W2040897483 cites W2024734780 @default.
• W2040897483 cites W2028528516 @default.
• W2040897483 cites W2033495952 @default.
• W2040897483 cites W2037473480 @default.
• W2040897483 cites W2041582506 @default.
• W2040897483 cites W2064612710 @default.
• W2040897483 cites W2064942594 @default.
• W2040897483 cites W2066699299 @default.
• W2040897483 cites W2069104086 @default.
• W2040897483 cites W2075299098 @default.
• W2040897483 cites W2081690388 @default.
• W2040897483 cites W2084841658 @default.
• W2040897483 cites W2085791091 @default.
• W2040897483 cites W2085931628 @default.
• W2040897483 cites W2110555897 @default.
• W2040897483 cites W2114972602 @default.
• W2040897483 cites W2129390376 @default.
• W2040897483 cites W2134394781 @default.
• W2040897483 cites W2135240540 @default.
• W2040897483 cites W2139305314 @default.
• W2040897483 cites W2143836761 @default.
• W2040897483 cites W333518702 @default.
• W2040897483 cites W4245342282 @default.
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