The role of tryptophan residues in Escherichia coli arginyl-tRNA synthetase

Biochimica et Biophysica Acta 1387 (1998) 136^142

The role of tryptophan residues in Escherichia coli arginyl-tRNA synthetase Qing-shuo Zhang, En-duo Wang *, Ying-lai Wang State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Academia Sinica, 320 Yue-yang Road, Shanghai 200031, China Received 31 March 1998; accepted 14 May 1998

Abstract The effect of N-bromosuccinimide (NBS) on the activity of Escherichia coli arginyl-tRNA synthetase (ArgRS) was studied. The results showed that only one tryptophan residue was easy of access to the reagent and was closely related to enzyme activity. When all the five tryptophan residues in ArgRS were changed via site-directed mutagenesis singly into Ala, the aminoacylation activity of the Trp162 mutated enzyme decreased seriously, while the other four mutant enzymes retained almost the same activity as the native one. The oxidation of the five mutant enzymes with NBS demonstrated that only the mutation of Trp162 resulted in the loss of sensitivity to the reagent. These results strongly suggest that Trp162 is more accessible to NBS and is related to enzyme activity. Furthermore, the far-UV CD spectroscopy of the mutant enzyme ArgRS162WA showed little change in its secondary structure. Finally, studies on the kinetics of the mutant enzyme ArgRS162WA in aminoacylation reaction showed that the reduction in activity could be attributed to the decrease in the values of kcat and kcat /Km for arginine. The thermodynamic calculation indicates that this mutation causes a decrease of the binding energy by 2.7 kJ/mol. Our data suggest that Trp162 is involved in the binding of arginine and in the transition state stabilization. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Arginyl-tRNA synthetase; Tryptophan; Site-directed mutagenesis

1. Introduction Aminoacyl-tRNA synthetase (aaRS) catalyzes the esteri¢cation of amino acid to the hydroxyl group at the 3P-end of its cognate transfer RNA [1]. Escherichia coli arginyl-tRNA synthetase (EC 6.1.1.19), which is a single peptide enzyme consisting of 577 amino acid residues, has been the focus of study for many years because of its interesting mechanism [2]. Arginyl-tRNA synthetase like glutamyl- and glu-

* Corresponding author. Fax: +86 (21) 6433-8357; E-mail: [email protected]

taminyl-tRNA synthetases requires the cognate tRNA to catalyze the ATP-PPi exchange reaction and is very di¡erent from the other aminoacyltRNA synthetases [3,4]. Tryptophan residues are involved in the active sites of many proteins, such as prothrombin [5], inorganic pyrophosphatase [6], polyphosphate/ATP glucokinase [7]. It has also been reported that a highly conserved single tryptophan residue in Bacillus subtilis tryptophanyl-tRNA synthetase is essential both in vitro and in vivo for the function of the enzyme [8]. E. coli arginyl-tRNA synthetase has ¢ve tryptophan residues: Trp162 , Trp172 , Trp228 , Trp349 and Trp446 . In order to investigate their role

0167-4838 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 8 ) 0 0 1 1 5 - 0

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in the enzymatic reaction, we used NBS to obtain the site-speci¢c chemical modi¢cation of these residues [9]. The results from chemical modi¢cation indicated that one particular tryptophan of the ¢ve was more accessible to the reagent and was related to enzyme activity. Then, we used site-directed mutagenesis for further investigation. The mutated argS genes were inserted into pUC18 vectors for overproduction of these mutant enzymes. This allowed an e¤cient puri¢cation of the mutant enzymes and a more thorough analysis of their properties and kinetics. 2. Materials and methods 2.1. Materials NBS was purchased from Sigma. tRNAArg was 2 puri¢ed to more than 90% homogeneity through a DEAE-Sepharose CL-6B and a BD-cellulose column from the E. coli strain TG1 containing a pTrc99Bderived plasmid, which could overproduce tRNAArg 2 . The argS gene of E. coli encoding native ArgRS was carried in the bacteriophage M13mp18. The plasmid pUC18 was used as a cloning vector to overexpress either the wild type or the mutated argS genes. 2.2. NBS titration Experiments were performed according to Spande and Witkop [10]. In a 1 cm silica cuvette was placed 1 ml of 100 mM potassium phosphate bu¡er, pH 7.0, containing 1 mg of the desired protein. After the initial optical density at 280 nm had been recorded, 10 Wl of a 10 mM aqueous solution of NBS was added to the cuvette until there was no further decrease in absorbance at 280 nm. The spectra were obtained as soon as possible after the addition of NBS. The minimum optical density was recorded and corrected for the volume increase due to the added reagent. The number of tryptophan residues titrated per mole was calculated employing the formula of Spande and Witkop [10]. Readings were made at 23^25³C in a Beckman model DU7400 spectrophotometer and the control solution contained the same bu¡er, but no enzymes.

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2.3. Site-directed mutagenesis and expression of argS gene Synthetic oligonucleotides WP1, WP2, WP3, WP4 and WP5 were used to convert the codons for Trp162 , Trp172 , Trp228 , Trp349 and Trp446 on the E. coli argS gene to those of Ala, respectively. Their sequences were: WP1: 5P-GAACTGAGTGCCCGCGTCGCCGAC-3P; WP2: 5P-GCTGCTTTTCCAGCGCTGCAATCAGCATACC-3P; WP3: 5P-GACCAGTTTGCGCGCCATCTCGCGG-3P; WP4: 5P-GGACGATCGCCGCTGCCTGCATCAGG-3P; WP5: 5P-CAGCATGTTGTCCGCGTCGAAGATGTAGTC-3P. The mutagenesis procedure developed by Kunkel [11] was used in this study. The mutants of argS con¢rmed by sequencing DNA were recombined with pUC18 and transformed into the E. coli strain TG1 for high expression.

2.4. Puri¢cation of the mutant arginyl-tRNA synthetase The disruption of cells was carried out according to the method of Eriani et al. [12]. The supernatant was applied to DEAE-Sepharose CL-6B equilibrated with bu¡er I (20 mM potassium phosphate bu¡er, pH 7.5). A continuous ionic strength gradient from 20 to 500 mM potassium phosphate, pH 7.5, was employed and the fraction containing ArgRS activity was collected and precipitated by adding ammonium sulfate to 75% saturation. The partially puri¢ed ArgRS was desalted by Sephadex G-25 and then applied to Blue Sepharose CL-6B equilibrated with bu¡er I. The same continuous ionic strength gradient was employed and the main fraction was collected. The enzyme preparation was concentrated and then dialyzed against bu¡er I containing 0.5 mM DTT, 0.2 mM PMSF, and 50% glycerol. This procedure su¤ced to yield an enzyme with more than 95% homogeneity.

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2.5. Determination of the aminoacylation activity The procedure was similar to the method of Lin et al [13]. A reaction mixture of 50 Wl contained 50 mM Tris-HCl, pH 7.4, 8 mM MgCl2 , 4 mM ATP, 80 mM KCl, 0.5 mM DTT, 0.1 mM EDTA, 20.5 mg/ml total tRNA (corresponding to a tRNAArg content of 534 2 WM, puri¢ed from the E. coli strain containing the gene of tRNAArg in its plasmid), and 0.1 mM 2 14 [ C]arginine (25 WCi/Wmol). One unit was de¢ned as the amount of enzyme which charged 1 nmol of tRNAArg in 1 min under the above conditions. Enzymatic assay was conducted at 37³C. 2.6. Kinetics The Km values for arginine and tRNAArg were determined under the conditions similar to the determination of the aminoacylation activity, except that the concentrations of the corresponding substrates were varied. When the Km value for ATP was measured, the concentration of Mg2‡ varied with that of ATP, keeping an excess Mg2‡ concentration of 4 mM. tRNAArg with a charging capacity of 1600 2 pmol/A260 unit (puri¢ed from the FPLC) was used. The Hanes^Woolf plot [14] was employed for data processing. 2.7. Fluorescence titration The interaction of the enzyme and arginine was studied by £uorescence titration. One milliliter of solution contained 100 Wg (1.5 WM) of native or mutant enzyme, 50 mM Tris-HCl (pH 7.5), 8 mM MgCl2 , and 0.2 mM DTT. After aliquots (1 Wl) of arginine solution (10 mM) were added into it, the £uorescence was assayed by a Hitachi F-4010 Fluorescence Spectrophotometer. Excitation and emission wavelengths were 295 and 350 nm with both bandwidths of 5 nm, respectively. Because of the excess substrate concentration ([S]E[E]0 , [E]0 denoted the input enzyme concentration), a simpli¢ed linear relation was used for data processing: vF = 3Kd vF/ [S]+vFr , where the symbols vF, vFr , and Kd referred to the change in £uorescence at the substrate concentration [S], to the £uorescence change when all enzyme molecules were complexed with substrates, and to the dissociation constant of the en-

zyme^substrate complex, respectively [15]. Appropriate corrections were made for volume changes inside the cuvette. All measurements were made at 22^ 25³C. 2.8. Circular dichroism spectroscopy Circular dichroism spectra were accumulated on a JAPAN Jasco-500A CD spectrometer. Protein samples were placed in a 5 mm path length cuvette. Spectra were averaged over ¢ve scans. 2.9. Protein concentration determination For all experiments, a modi¢ed method of Lowry et al. [16] by Bensadoun and Weinstein [17] was applied to determine the protein concentration. 3. Results 3.1. Oxidation of the arginyl-tRNA synthetase with NBS The results of the titration of ArgRS in potassium phosphate bu¡er (100 mM, pH 7.0) are graphically presented in Fig. 1. When ArgRS in 100 mM potassium phosphate solution (pH 7.0) was treated with NBS, only one of the ¢ve tryptophan residues was oxidized, although 10.4 mol of NBS per mol of protein was consumed at this point. Oxidized enzymes

Fig. 1. Oxidization of tryptophan residues of ArgRS at pH 7.0: F, no. of tryptophan residues oxidized; b, loss of activity. These data were obtained within 5 min of the initiation of the reaction.

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Table 1 Arginylation of tRNA by ArgRS and its mutants Enzyme

Relative activity (%)

Native ArgRS ArgRS162WA ArgRS172WA ArgRS228WA ArgRS349WA ArgRS446WA

100 23.3 102 95.5 91.4 98.7

Enzymatic units per milligram protein were measured. One unit is de¢ned as the amount of enzyme which charges 1 nmol of tRNAArg in 1 min under the conditions described in Section 2. The activity value for the native ArgRS is 22 500 U/mg. Accuracy is þ 5% for relative activities of mutant enzymes (standard error).

displayed only 13% of the original activity. The results suggest that one particular tryptophan in ArgRS is more accessible to the reagent and is related to enzyme activity. 3.2. Arginylation of tRNAArg by the native ArgRS and ¢ve mutant enzymes After the ¢ve mutated argS genes were cloned into pUC18 and were expressed in TG1, the native and ¢ve mutant enzymes (ArgRS162WA, ArgRS172WA, ArgRS228WA, ArgRS349WA and ArgRS446WA) were puri¢ed and assayed for tRNA arginylation, respectively. Puri¢cation of the native or mutant ArgRS yielded the homogeneous enzymes shown in Fig. 2. Relative activities of mutant enzymes are shown in Table 1. Only ArgRS162WA displayed decreased enzymatic activity. It had only 23.3% of activity of native enzyme. These data suggest that Trp162 in ArgRS is related to the enzymatic activity. It is probably the very tryptophan residue sensitive to NBS. 3.3. Oxidization of ¢ve mutant enzymes with NBS Studies on the oxidizability of tryptophan with NBS in the ¢ve mutated enzymes showed that ArgRS162WA displayed little reactivity while all the other four mutated enzymes, like the native enzyme, had a tryptophan residue accessible to NBS. This result con¢rms our expectation that Trp162 is more accessible to NBS. We can conclude that

Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoretogram. Lane 1, molecular weight markers for SDS-PAGE ; lane 2, crude extract from TG1[pUC18-argS]; lane 3, after chromatography on DEAE-Sepharose; lane 4, after chromatography on Blue Sepharose CL6B.

Trp162 may lie in a more exposed environment on the surface of ArgRS. 3.4. Steady-state kinetics of ArgRS162WA The kinetic constants of the arginylation reaction for ArgRS162WA and ArgRS were presented in Table 2. Km for arginine and kcat of ArgRS162WA both decreased in comparison with the counterparts of the native ArgRS. These changes of ArgRS162WA led to a lower kcat /Km . The mutation causes a loss of Table 2 Kinetic constants for the aminoacylation reaction by ArgRS and ArgRS162WA Km

KArg (WM) KATP (mM) KtRNA (WM) Vmax (U/mg) kcat (s31 ) kcat /KArg (106 s31 M31 ) vvGg (kJ mol31 )

ArgRS162WA

ArgRS

7.3 0.84 2.3 5200 5.6 0.77 2.7

12 0.90 2.5 24 000 26 2.2 0

In these experiments, enzyme concentrations were normalized to 0.56 Wg/ml (8.7 nM) of ArgRS and 2.6 Wg/ml (40 nM) of ArgRS162WA. First-order (kcat ) and second-order (kcat /Km ) rate constants were calculated from apparent Vmax and Vmax /Km values according to the exact molecular weight of 64 692. vvGg , referring to the decrease of the binding energy of the mutant enzyme relative to the native one, was calculated from the relation [18]: vvGg = 3RTln[(kcat /Km )mut/(kcat /Km )wt].

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indicate that ArgRS162WA has an enhanced a¤nity with arginine. 3.6. Far-UV CD spectra The CD spectrum of ArgRS162WA is shown in Fig. 4. The change of Trp162 to Ala162 induced very little change in the appearance of the CD spectrum indicating little change in its secondary structure. 4. Discussion Fig. 3. Titration of the ArgRS and ArgRS162WA £uorescence by arginine. The dissociation constant of the complex between the enzyme and arginine was determined by the slope.

2.7 kJ/mol of binding energy calculated from the relation: vvGg = 3RTln[(kcat /Km )mut/(kcat /Km )wt] [18]. As for the other two substrates, ATP and tRNAArg , the Km values of the mutant enzyme showed little change. 3.5. Fluorescence titration The binding of arginine was studied by £uorescence titration. In the absence of other substrates, the titration of the ArgRS £uorescence by arginine and that of the ArgRS162WA £uorescence gave the dissociation constant values (KArg ) of 75 and 29 WM, respectively (Fig. 3). These data, in accordance with the changes of kinetic constants of ArgRS162WA,

Fig. 4. Circular dichroism spectra of ArgRS162WA and native ArgRS. The protein concentration was 0.1 mg/ml containing 50 mM Tris-HCl (pH 7.5) and 10 mM MgCl2 . The spectra were recorded at ambient temperature (V16³C).

In this study, N-bromosuccinimide was used to obtain the site-speci¢c chemical modi¢cation of tryptophan residue in ArgRS. When ArgRS in 100 mM potassium phosphate solution (pH 7.0) was treated with NBS, only one of the ¢ve tryptophan residues was oxidized. Oxidized enzyme displayed only 13% of the initial activity. The experiment of site-directed mutagenesis of tryptophan residues showed that only ArgRS162WA, whose Trp162 had been changed to Ala, exhibited a serious decrease of aminoacylation activity relative to native ArgRS. At the same time, ArgRS162WA was not sensitive toward NBS compared with the native enzyme, while the other four mutated enzymes displayed the same reactivity towards NBS as the native enzyme. Therefore, we can conclude that Trp162 in ArgRS is the very tryptophan residue sensitive to NBS. Trp162 may lie in a more exposed environment on the surface of ArgRS and is closely related to enzyme activity. The other four tryptophan residues are not conserved and not essential to enzyme activity. Aminoacyl-tRNA synthetases can be divided into two classes of ten members each. ArgRS is classi¢ed as a Class I synthetase, which active site should be folded in a manner known as the Rossmann fold [19]. Sequence alignments show that Trp162 is located in this domain [20]. According to the result from prediction of secondary structure, Trp162 is located in the loop just after the second helix of the Rossmann fold. For further investigations, we studied properties of ArgRS162WA to determine the role of Trp162 in ArgRS. As it was shown above, ArgRS162WA displayed a reduced Km of the arginylation reaction for arginine and a reduced KArg in £uorescence titration. This indicates that ArgRS162-

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WA has an enhanced a¤nity with arginine. However, the mutant enzyme exhibited much lower ¢rst-order (kcat ) and second-order (kcat /Km ) rate constants than the native one. kcat /Km is related to the total binding energy of an enzyme and a substrate [18]. Binding energy can be used to lower the activation energy of the chemical step and to stabilize the transition state. The replacement of Trp162 by Ala causes a loss of 2.7 kJ/mol of binding energy. For this reason, ArgRS162WA is a poorer physiological catalyst despite the improvement in arginine binding. In conclusion, Trp162 is involved in the binding of arginine and in the transition state stabilization. Previous investigations have revealed that the two ArgRS species from E. coli and Chinese hamster ovary cells (CHO) show a high level of identity between their primary structures [21]. In particular, two segments of 15 amino acids, from Ala125 to Gly139 and from Asn157 to Ala171 of E. coli ArgRS, are shared by the two enzymes. According to the known 3D-crystal structure of several Class I aaRS, the two conserved segments should be located in the active site of ArgRS [22^25]. The ¢rst segment contains the HIGH signature sequence, characteristic of class I aaRS [1,26]. The HIGH sequence is thought to be involved in the binding of ATP, the common substrate of these enzymes [27]. The role of the second segment NHVGDWGTQFGMLIA remains to be determined. It is present only in ArgRS. Trp162 is located near the center of this homologous sequence. Our data indicate that Trp162 is probably involved in the binding of arginine and in the transition state stabilization. Therefore, we suggest that this segment may interact with arginine, a common substrate of the two ArgRS species. Acknowledgements

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9] [10]

[11]

[12]

[13]

[14]

This work was supported by a grant from the National Natural Science Foundation of China.

[15]

References

[16]

[1] P. Schimmel, Aminoacyl-tRNA synthetases: general scheme of structure^function relationships in the polypeptides and

[17]

141

recognition of transfer RNAs, Annu. Rev. Biochem. 56 (1987) 125^158. G. Eriani, G. Dirheimer, J. Ganglo¡, Isolation and characterization of the gene coding for Escherichia coli arginyltRNA synthetase, Nucleic Acids Res. 17 (1989) 5725^ 5736. A.H. Mehler, S.K. Mitra, The activation of arginyl transfer ribonucleic acid synthetase by transfer ribonucleic acid, J. Biol. Chem. 242 (1967) 5495^5499. T.S. Papas, A. Peterkofsky, A random sequential mechanism for arginine transfer ribonucleic acid synthetase of Escherichia coli, Biochemistry 11 (1972) 4602^4608. W.K. Stevens, M.E. Nesheim, Structural changes in the protease domain of prothrombin upon activation as assessed by N-bromosuccinimide modi¢cation of tryptophan residues in prethrombin-2 and thrombin, Biochemistry 32 (1993) 2787^ 2794. S.I. Kaneko, T. Ichiba, N. Hirano, A. Hachimori, Modi¢cation of a single tryptophan of the inorganic pyrophosphatase from thermophilic bacterium PS-3: possible involvement in its substrate binding, Biochem. Biophys. Acta 1077 (1991) 281^284. P.C. Hsieh, B.C. Shenoy, F.C. Haase, J.E. Jentoft, N.F.B. Phillips, Involvement of tryptophan(s) at the active site of polyphosphate/ATP glucokinase from Mycobacterium tuberculosis, Biochemistry 32 (1993) 6243^6249. K. Chow, H. Xue, W. Shi, J.T. Wong, Mutational identi¢cation of an essential tryptophan in tryptophanyl-tRNA synthetase of Bacillus subtilis, J. Biol. Chem. 267 (1992) 9146^ 9149. R.L. Lundblad, Techniques in Protein Modi¢cation, CRC Press, Boca Raton, FL, 1995. T.F. Spande, B. Witkop, Determination of the tryptophan content of proteins with N-bromosuccinimide, Methods Enzymol. 11 (1967) 498^505. T.A. Kunkel, Rapid and e¤cient site-speci¢c mutagenesis without phenotypic selection, Proc. Natl. Acad. Sci. USA 82 (1985) 488^492. G. Eriani, G. Dirheimer, J. Ganglo¡, Structure^function relationship of arginyl-tRNA synthetase from Escherichia coli: isolation and characterization of the argS mutation MA5002, Nucleic Acids Res. 18 (1990) 1475^1479. S.X. Lin, J.P. Shi, X.D. Cheng, Y.L. Wang, Arginyl-tRNA synthetase from Escherichia coli: puri¢cation by a¤nity chromatography, properties, and steady-state kinetics, Biochemistry 27 (1988) 6343^6348. I.H. Segel, Biochemical Calculations, Wiley, New York, 1975. E. Holler, E.L. Bennett, M. Calvin, 2-p-Toluidinylnaphthalene-6-sulfonate, a £uorescent reporter group for L-isoleucyltRNA synthetase, Biochem. Biophys. Res. Commun. 45 (1971) 409^415. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265^275. A. Bensadoun, D. Weinstein, Assay of proteins in the pres-

BBAPRO 35696 27-8-98

142

[18]

[19]

[20] [21]

[22]

Q.-s. Zhang et al. / Biochimica et Biophysica Acta 1387 (1998) 136^142 ence of interfering materials, Anal. Biochem. 70 (1976) 241^ 250. A.J. Wilkinson, A.R. Fersht, D.M. Blow, G. Winter, Sitedirected mutagenesis as a probe of enzyme structure and catalysis : tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation, Biochemistry 22 (1983) 3581^3586. M.G. Rossmann, D. Moras, K.W. Olsen, Chemical and biological evolution of a nucleotide binding domain, Nature 250 (1974) 194^199. M. Delarue, D. Moras, The aminoacyl-tRNA synthetase family: modules at work, Bioessays 15 (1993) 675^687. M. Lazard, M. Mirande, Cloning and analysis of a cDNA encoding mammalian arginyl-tRNA synthetase, a component of the multisynthetase complex with a hydrophobic N-terminal extension, Gene 132 (1993) 237^245. P. Brick, T.N. Bhat, D.M. Blow, Structure of tyrosyl-tRNA î resolution: interaction of the synthetase re¢ned at 2.3 A enzyme with the tyrosyl adenylate intermediate, J. Mol. Biol. 208 (1989) 83^98.

[23] M.A. Rould, J.J. Perona, D. So«ll, T. Steitz, Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNAGln î resolution, Science 246 (1989) 1135^1142. and ATP at 2.8 A [24] M.A. Rould, J.J. Perona, T. Steitz, Structural basis of anticodon loop recognition by glutaminyl-tRNA synthetase, Nature 352 (1991) 213^218. [25] S. Brunie, C. Zelwer, J.L. Risler, Crystallographic study at î resolution of the interaction of methionyl-tRNA syn2.5 A thetase from Escherichia coli with ATP, J. Mol. Biol. 216 (1990) 411^424. [26] G. Eriani, M. Delarue, O. Poch, J. Ganglo¡, D. Moras, Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs, Nature 347 (1990) 203^206. [27] T.A. Webster, H. Tsai, M. Kula, G.A. Mackie, P. Schimmel, Speci¢c sequence homology and three dimensional structure of an aminoacyl transfer RNA synthetase, Science 226 (1984) 1315^1317.

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