Species-specific Differences in Amino Acid Editing by Class II Prolyl-tRNA Synthetase
2001; Elsevier BV; Volume: 276; Issue: 33 Linguagem: Inglês
10.1074/jbc.m104761200
ISSN1083-351X
AutoresPenny J. Beuning, Karin Musier‐Forsyth,
Tópico(s)Mitochondrial Function and Pathology
ResumoAminoacyl-tRNA synthetases are a family of enzymes responsible for ensuring the accuracy of the genetic code by specifically attaching a particular amino acid to their cognate tRNA substrates. Through primary sequence alignments, prolyl-tRNA synthetases (ProRSs) have been divided into two phylogenetically divergent groups. We have been interested in understanding whether the unusual evolutionary pattern of ProRSs corresponds to functional differences as well. Previously, we showed that some features of tRNA recognition and aminoacylation are indeed group-specific. Here, we examine the species-specific differences in another enzymatic activity, namely amino acid editing. Proofreading or editing provides a mechanism by which incorrectly activated amino acids are hydrolyzed and thus prevented from misincorporation into proteins. "Prokaryotic-like" Escherichia coli ProRS has recently been shown to be capable of misactivating alanine and possesses both pretransfer and post-transfer hydrolytic editing activity against this noncognate amino acid. We now find that two ProRSs belonging to the "eukaryotic-like" group exhibit differences in their hydrolytic editing activity. Whereas ProRS from Methanococcus jannaschii is similar to E. coli in its ability to hydrolyze misactivated alanine via both pretransfer and post-transfer editing pathways, human ProRS lacks these activities. These results have implications for the selection or design of antibiotics that specifically target the editing active site of the prokaryotic-like group of ProRSs. Aminoacyl-tRNA synthetases are a family of enzymes responsible for ensuring the accuracy of the genetic code by specifically attaching a particular amino acid to their cognate tRNA substrates. Through primary sequence alignments, prolyl-tRNA synthetases (ProRSs) have been divided into two phylogenetically divergent groups. We have been interested in understanding whether the unusual evolutionary pattern of ProRSs corresponds to functional differences as well. Previously, we showed that some features of tRNA recognition and aminoacylation are indeed group-specific. Here, we examine the species-specific differences in another enzymatic activity, namely amino acid editing. Proofreading or editing provides a mechanism by which incorrectly activated amino acids are hydrolyzed and thus prevented from misincorporation into proteins. "Prokaryotic-like" Escherichia coli ProRS has recently been shown to be capable of misactivating alanine and possesses both pretransfer and post-transfer hydrolytic editing activity against this noncognate amino acid. We now find that two ProRSs belonging to the "eukaryotic-like" group exhibit differences in their hydrolytic editing activity. Whereas ProRS from Methanococcus jannaschii is similar to E. coli in its ability to hydrolyze misactivated alanine via both pretransfer and post-transfer editing pathways, human ProRS lacks these activities. These results have implications for the selection or design of antibiotics that specifically target the editing active site of the prokaryotic-like group of ProRSs. isoleucyl-tRNA synthetase connective polypeptide 1 valyl-tRNA synthetase leucyl-tRNA synthetase threonyl-tRNA synthetase prolyl-tRNA synthetase phenylalanyl-tRNA synthetase Specific aminoacylation of tRNAs by aminoacyl-tRNA synthetases is critical for the accurate translation of the genetic code. This is accomplished in a two-step process. In the first step, the cognate amino acid is activated with ATP to form the aminoacyl-adenylate. In the second step, the amino acid is transferred to the 3′-end of the cognate tRNA (1Ibba M. Söll D. Annu. Rev. Biochem. 2000; 69: 617-650Crossref PubMed Scopus (1115) Google Scholar). The aminoacyl-tRNA synthetases must ensure that the relationship between anticodon and amino acid is faithfully maintained (1Ibba M. Söll D. Annu. Rev. Biochem. 2000; 69: 617-650Crossref PubMed Scopus (1115) Google Scholar). It is expected that the selection of the correct amino acid is more error-prone than the selection of the cognate tRNA isoacceptor group, because the amino acid is a much smaller molecule with fewer distinguishing features (2Fersht A.R. Proc. R. Soc. Lond. B Biol. Sci. 1981; 212: 351-379Crossref PubMed Scopus (70) Google Scholar, 3Jakubowski H. Goldman E. Microbiol. Rev. 1992; 56: 412-429Crossref PubMed Google Scholar). Misactivation of amino acids can be corrected in several ways. In pretransfer editing, the noncognate aminoacyl-adenylate is hydrolyzed by the synthetase in an ATP-dependent manner, and in many cases this activity is stimulated by the presence of the cognate tRNA (3Jakubowski H. Goldman E. Microbiol. Rev. 1992; 56: 412-429Crossref PubMed Google Scholar). In post-transfer editing, a mischarged tRNA is deacylated in an ATP-independent manner (3Jakubowski H. Goldman E. Microbiol. Rev. 1992; 56: 412-429Crossref PubMed Google Scholar). Synthetases may possess one or both types of editing activities and, in some cases, lack an editing function altogether. In general, amino acid editing has been less well characterized than tRNA recognition. However, there are several well studied editing systems, especially among class I synthetases. For example, isoleucyl-tRNA synthetase (IleRS)1 misactivates valine and hydrolyzes misactivated Val-AMP as well as mischarged Val-tRNAIle (4Baldwin A.N. Berg P. J. Biol. Chem. 1966; 241: 839-845Abstract Full Text PDF PubMed Google Scholar, 5Eldred E.W. Schimmel P.R. J. Biol. Chem. 1972; 247: 2961-2964Abstract Full Text PDF PubMed Google Scholar, 6Schmidt E. Schimmel P. Science. 1994; 264: 265-267Crossref PubMed Scopus (138) Google Scholar). In IleRS, a subclass Ia-specific insertion domain, the so-called connective polypeptide 1 (CP1), has been shown to be responsible for post-transfer editing (7Schmidt E. Schimmel P. Biochemistry. 1995; 34: 11204-11210Crossref PubMed Scopus (78) Google Scholar, 8Lin L. Hale S.P. Schimmel P. Nature. 1996; 384: 33-34Crossref PubMed Scopus (92) Google Scholar, 9Lin L. Schimmel P. Biochemistry. 1996; 35: 5596-5601Crossref PubMed Scopus (38) Google Scholar, 10Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Science. 1998; 280: 578-582Crossref PubMed Scopus (316) Google Scholar). The CP1 domain is highly conserved among IleRSs, including those fromEscherichia coli, yeast, and humans (7Schmidt E. Schimmel P. Biochemistry. 1995; 34: 11204-11210Crossref PubMed Scopus (78) Google Scholar, 11Hendrickson T.L. Nomanbhoy T.K. Schimmel P. Biochemistry. 2000; 39: 8180-8186Crossref PubMed Scopus (45) Google Scholar). Accordingly, the editing activity of IleRS is present in all species examined to date (5Eldred E.W. Schimmel P.R. J. Biol. Chem. 1972; 247: 2961-2964Abstract Full Text PDF PubMed Google Scholar, 6Schmidt E. Schimmel P. Science. 1994; 264: 265-267Crossref PubMed Scopus (138) Google Scholar, 10Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Science. 1998; 280: 578-582Crossref PubMed Scopus (316) Google Scholar, 12Freist W. Pardowitz I. Cramer F. Biochemistry. 1985; 24: 7014-7023Crossref PubMed Scopus (41) Google Scholar, 13Silvian L.F. Wang J. Steitz T.A. Science. 1999; 285: 1074-1077Crossref PubMed Scopus (360) Google Scholar). Two other class Ia enzymes, ValRS and LeuRS, also possess editing functions (8Lin L. Hale S.P. Schimmel P. Nature. 1996; 384: 33-34Crossref PubMed Scopus (92) Google Scholar, 9Lin L. Schimmel P. Biochemistry. 1996; 35: 5596-5601Crossref PubMed Scopus (38) Google Scholar, 14Chen J.-F. Guo N.-N. Li T. Wang E.-D. Wang Y.-L. Biochemistry. 2000; 39: 6726-6731Crossref PubMed Scopus (116) Google Scholar, 15Mursinna R.S. Lincecum Jr., T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar). The CP1 domain of both of these enzymes has been shown to be responsible for editing threonine in the case of ValRS and isoleucine and methionine in the case of LeuRS (8Lin L. Hale S.P. Schimmel P. Nature. 1996; 384: 33-34Crossref PubMed Scopus (92) Google Scholar, 9Lin L. Schimmel P. Biochemistry. 1996; 35: 5596-5601Crossref PubMed Scopus (38) Google Scholar, 14Chen J.-F. Guo N.-N. Li T. Wang E.-D. Wang Y.-L. Biochemistry. 2000; 39: 6726-6731Crossref PubMed Scopus (116) Google Scholar, 15Mursinna R.S. Lincecum Jr., T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar, 16Fukai S. Nureki O. Sekine S.-I. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Similar to IleRS, the CP1 domains of ValRS and LeuRS are highly conserved (9Lin L. Schimmel P. Biochemistry. 1996; 35: 5596-5601Crossref PubMed Scopus (38) Google Scholar, 15Mursinna R.S. Lincecum Jr., T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar), and editing activity is present in a range of species examined thus far (9Lin L. Schimmel P. Biochemistry. 1996; 35: 5596-5601Crossref PubMed Scopus (38) Google Scholar, 14Chen J.-F. Guo N.-N. Li T. Wang E.-D. Wang Y.-L. Biochemistry. 2000; 39: 6726-6731Crossref PubMed Scopus (116) Google Scholar, 15Mursinna R.S. Lincecum Jr., T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar,17Fersht A.R. Kaethner M.M. Biochemistry. 1976; 15: 3342-3346Crossref PubMed Scopus (180) Google Scholar, 18Igloi G.L. von der Haar F. Cramer F. Biochemistry. 1978; 17: 3459-3468Crossref PubMed Scopus (42) Google Scholar, 19Fersht A.R. Dingwall C. Biochemistry. 1979; 18: 1238-1245Crossref PubMed Scopus (61) Google Scholar, 20Fersht A.R. Dingwall C. Biochemistry. 1979; 18: 2627-2631Crossref PubMed Scopus (121) Google Scholar, 21Jakubowski H. Biochemistry. 1980; 19: 5071-5078Crossref PubMed Scopus (29) Google Scholar, 22Englisch S. Englisch U. von der Haar F. Cramer F. Nucleic Acids Res. 1986; 14: 7529-7539Crossref PubMed Scopus (78) Google Scholar). Some class II synthetases have also been shown to edit noncognate amino acids. Recent structural and biochemical work showed that the N-terminal domain of E. coli threonyl-tRNA synthetase (ThrRS) is responsible for editing mischarged Ser-tRNAThr(23Dock-Bregeon A. Sankaranarayanan R. Romby P. Caillet J. Springer M. Rees B. Francklyn C. Ehresmann C. Moras D. Cell. 2000; 103: 877-884Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). This class II synthetase uses an active site Zn2+ ion to prevent misactivation of isosteric, but hydrophobic, valine (24Sankaranarayanan R. Dock-Bregeon A.-C. Rees B. Bovee M. Caillet J. Romby P. Francklyn C.S. Moras D. Nat. Struct. Biol. 2000; 7: 461-465Crossref PubMed Scopus (142) Google Scholar). However, this Zn2+ ion cannot prevent the activation of serine, whose hydroxyl group is able to bind to the metal ion. Thus, in the absence of the N-terminal editing domain, the misactivated seryl-adenylate is charged onto tRNAThr (24Sankaranarayanan R. Dock-Bregeon A.-C. Rees B. Bovee M. Caillet J. Romby P. Francklyn C.S. Moras D. Nat. Struct. Biol. 2000; 7: 461-465Crossref PubMed Scopus (142) Google Scholar). The N-terminal domain is highly conserved among ThrRSs from all species except archaebacteria, and to date, ThrRSs from both E. coliand yeast have been shown to edit serine (23Dock-Bregeon A. Sankaranarayanan R. Romby P. Caillet J. Springer M. Rees B. Francklyn C. Ehresmann C. Moras D. Cell. 2000; 103: 877-884Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 25Freist W. Sternbach H. Cramer F. Eur. J. Biochem. 1994; 220: 745-752Crossref PubMed Scopus (10) Google Scholar). We recently showed that another class II synthetase, E. coli prolyl-tRNA synthetase (ProRS), misactivates noncognate amino acids, including alanine (26Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8920Crossref PubMed Scopus (121) Google Scholar). The side chain of alanine is smaller than that of proline and could easily be accommodated in the amino acid binding pocket of ProRS. Indeed, this enzyme possesses both pre- and post-transfer editing activity against alanine, because it has been shown to hydrolyze misactivated Ala-AMP in a tRNA-independent fashion and to rapidly deacylate a mischarged Ala-tRNAPro variant (26Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8920Crossref PubMed Scopus (121) Google Scholar). The family of ProRSs can be sorted into two phylogenetically distinct groups based on their primary structures (27Cusack S. Yaremchuk A. Krikliviy I. Tukalo M. Structure. 1998; 6: 101-108Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 28Stehlin C. Burke B. Yang F. Liu H. Shiba K. Musier-Forsyth K. Biochemistry. 1998; 37: 8605-8613Crossref PubMed Scopus (61) Google Scholar). The "prokaryotic-like" group, consisting of prokaryotic and putative eukaryotic mitochondrial enzymes, is characterized by a large insertion domain (185 amino acids in the case of E. coli ProRS) between motifs 2 and 3. In contrast, the "eukaryotic-like" group, consisting of ProRSs from eukarya, archaea, and a few bacteria, lacks the prokaryotic insertion domain but instead has a C-terminal extension that is missing in the prokaryotic-like group. Thus, the sorting of ProRSs into two distinct groups does not conform to the canonical phylogenetic division of bacterial and archaeal/eukaryal synthetases (28Stehlin C. Burke B. Yang F. Liu H. Shiba K. Musier-Forsyth K. Biochemistry. 1998; 37: 8605-8613Crossref PubMed Scopus (61) Google Scholar). A recent structure-based phylogeny of ProRSs reinforced the notion that there are major differences between the canonical universal tree and ProRS (29Ribas de Pouplana, L., Brown, J. R., and Schimmel, P. (2001)J. Mol. Evol., in press.Google Scholar). In particular, a mixture of ProRS types is found even among closely related species of eubacteria, and lateral transfer within a canonical three-kingdom structure cannot account for the unusual positions of eubacterial and eukaryotic sequences. Using representative ProRSs from all three domains of life, we previously demonstrated species-specific differences in tRNA recognition (28Stehlin C. Burke B. Yang F. Liu H. Shiba K. Musier-Forsyth K. Biochemistry. 1998; 37: 8605-8613Crossref PubMed Scopus (61) Google Scholar, 30Burke B. Yang F., F., C. Stehlin C. Chan B. Musier-Forsyth K. Biochemistry. 2000; 39: 15540-15547Crossref PubMed Scopus (21) Google Scholar, 31Burke, B., Lipman, R. S. A., Shiba, K., Musier-Forsyth, K., and Hou, Y.-M. (2001) J. Biol. Chem., 276.Google Scholar). In particular, whereas anticodon recognition is important for ProRSs from both the prokaryotic-like and eukaryotic-like groups, base-specific acceptor stem recognition is critical only for the prokaryotic-like E. coli enzyme (32Liu H. Peterson R. Kessler J. Musier-Forsyth K. Nucleic Acids Res. 1995; 23: 165-169Crossref PubMed Scopus (53) Google Scholar). In contrast, base-specific acceptor stem discrimination is not an important feature of human ProRS, and similarly, the eukaryotic-likeMethanococcus jannaschii ProRS has only modest acceptor stem recognition (31Burke, B., Lipman, R. S. A., Shiba, K., Musier-Forsyth, K., and Hou, Y.-M. (2001) J. Biol. Chem., 276.Google Scholar). Thus, the segregation of ProRSs into two evolutionarily divergent groups is reflected in differences in tRNA acceptor stem recognition. Although the editing function of a prokaryotic ProRS has been described (26Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8920Crossref PubMed Scopus (121) Google Scholar), it is unknown whether this function is conserved throughout evolution. The prokaryotic-like insertion domain is similar in size to the CP1 editing domain present in class I IleRS and is a good candidate for an editing domain. However, this domain is missing in the eukaryotic-like group, and the unique C-terminal extension present only in the latter group bears little resemblance to the prokaryotic insertion. Thus, in this work, we chose to study both the human and the archaebacterial M. jannaschii ProRSs as representative eukaryotic-like enzymes. We show that only the archaebacterial synthetase, like the E. coli enzyme, possesses hydrolytic editing activity. Thus, the division of ProRSs into two distinct groups is not strictly correlated with the presence or the absence of amino acid editing functions. All amino acids were purchased from Sigma and were of the highest quality commercially available. Creation of mutant tRNA genes was accomplished using the QuikChange site-directed mutagenesis kit (Stratagene). RNA was transcribed fromBstN1-linearized plasmid using T7 RNA polymerase as described (28Stehlin C. Burke B. Yang F. Liu H. Shiba K. Musier-Forsyth K. Biochemistry. 1998; 37: 8605-8613Crossref PubMed Scopus (61) Google Scholar, 33Liu H. Yap L.-P. Musier-Forsyth K. J. Am. Chem. Soc. 1996; 118: 2523-2524Crossref Scopus (19) Google Scholar). Transcripts were gel purified on denaturing 12% polyacrylamide-TBE gels, eluted, and desalted using published conditions (34Liu H. Musier-Forsyth K. Biochemistry. 1994; 33: 12708-12714Crossref PubMed Scopus (46) Google Scholar). Bulk native E. coli tRNA was purchased from Sigma. Bulk native tRNA from M. jannaschii was a gift from Prof. Ya-Ming Hou (Thomas Jefferson University). Overexpression and purification of histidine-tagged E. coli, human, andM. jannaschii ProRS from E. coli cells were performed as described previously (31Burke, B., Lipman, R. S. A., Shiba, K., Musier-Forsyth, K., and Hou, Y.-M. (2001) J. Biol. Chem., 276.Google Scholar, 35Heacock D. Forsyth C.J. Shiba K. Musier-Forsyth K. Bioorganic Chem. 1996; 24: 273-289Crossref Scopus (108) Google Scholar). The ATP-PPiexchange reaction was performed according to the published conditions (35Heacock D. Forsyth C.J. Shiba K. Musier-Forsyth K. Bioorganic Chem. 1996; 24: 273-289Crossref Scopus (108) Google Scholar). For E. coli and human ProRS, the concentration of proline ranged from 0.05–2 mm, whereas with M. jannaschii ProRS the proline range was 0.001–0.5 mm. The alanine concentrations for all three enzymes ranged from 25 to 500 mm. The concentrations of cysteine assayed ranged from 10 to 100 mm. M. jannaschii ProRS concentrations were 1 nm for activation of proline and cysteine, and 61.5 nm for activation of other noncognate amino acids. Human ProRS concentrations were 1 nm for proline and 20 nm for noncognate amino acids. Assays with the E. coli and human enzymes were performed at 37 °C, whereas those with M. jannaschii ProRS were carried out at 60 °C. Kinetic parameters were determined from Lineweaver-Burk plots and represent the average of at least three determinations. ATP hydrolysis reactions were performed according to the published procedure (26Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8920Crossref PubMed Scopus (121) Google Scholar). Those reactions with tRNA contained 3–10 µm cognate tRNA. The reactions were initiated with enzyme to give the following final concentrations of ProRS: 0.9 µm (human), 0.5 µm (M. jannaschii), and 1–2 µm (E. coli). The assays with E. coli and human ProRS were performed either at room temperature or at 37 °C, whereas those with M. jannaschii ProRS were carried out at 60 °C. Aminoacylation assays were performed at room temperature according to published conditions (36Musier-Forsyth K. Scaringe S. Usman N. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 209-213Crossref PubMed Scopus (68) Google Scholar). When charged tRNA was isolated for deacylation assays, all of the amino acid present in the reaction was from a tritiated stock (55 Ci/mmol Ala or 90 Ci/mmol Pro). In some cases, inorganic pyrophosphatase (2 units/ml) was added to increase the extent of mischarging. The assays were first performed to determine the required time to reach plateau levels of aminoacylation. At the desired time, acetic acid was added to 1% final concentration to quench the reaction. The charged tRNA was purified by repeated phenol:CHCl3:isoamyl alcohol (50:48:2) extractions, followed by ethanol precipitation. Phenol was equilibrated against diethylpyrocarbonate-treated water. Charged tRNA was quantified by scintillation counting and stored at −20 °C in 50 mm KPO4, pH 5.0. Mischarging assays were carried out using the standard conditions, with the exception of the enzyme and tRNA concentrations. In these experiments, 5.0 µm tRNA was used along with the following concentrations of ProRS: 4.4 µm (M. jannaschii), 2.0 µm (human), and 5.3 µm (E. coli). Assays with E. coliand human ProRS were performed at room temperature, whereas assays withM. jannaschii ProRS were carried out at 60 °C. The deacylation reactions were performed as described (26Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8920Crossref PubMed Scopus (121) Google Scholar) and contained 0.1–1.0 µm charged tRNA in a final reaction volume of 70 µl. The reactions were carried out at 24 °C and were initiated with human ProRS (2.0 µm) or with NaOH (0.12 n) as a positive control. Negative controls were identical to these reactions except with enzyme omitted. The reactions with M. jannaschii ProRS (4.4 µm) were performed at 37 °C. At each time point, 10-µl aliquots were removed, and the reactions were quenched on Whatman 3MM filter pads, which had been presoaked with 5% trichloroacetic acid. The pads were immediately dropped into ice-cold 5% trichloroacetic acid and washed as described for aminoacylation assays (36Musier-Forsyth K. Scaringe S. Usman N. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 209-213Crossref PubMed Scopus (68) Google Scholar). All 20 amino acids were tested in the ATP-PPi exchange reaction for activation byE. coli, M. jannaschii, and human ProRS. All three enzymes activate select noncognate amino acids to varying extents. We previously found that E. coli ProRS activates alanine, with significantly decreased k cat and elevated K m (26Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8920Crossref PubMed Scopus (121) Google Scholar). The overall reduction in efficiency of activation of alanine relative to proline by the E. coli enzyme is ∼104-fold. Similary, human andM. jannaschii ProRSs activate alanine, albeit with ∼105-fold reduced efficiency relative to cognate proline (Table I). Therefore, based solely on the relative catalytic efficiency (k cat/K m) of alanine activation, both eukaryotic-like ProRSs examined in this study are approximately 1 order of magnitude more accurate in the initial selection of cognate amino acid than E. coli ProRS. All three enzymes activate other amino acids, most notably glycine, to a measurable extent (data not shown).Table IKinetic parameters for activation of proline and alanine by E. coli, M. jannaschii, and human ProRSProRS speciesAmino acidk catK mk cat/K mk cat/K m(relative)1-akcat/K mis relative to proline in each set, which was set at 1.0. Individual kinetic parameters are based on the average of three determinations.s−1mms−1mm−1E. coliProline1-bAs reported in Ref. 26.70 ± 250.25 ± 0.0352801Alanine1-bAs reported in Ref. 26.1.7 ± 0.56140 ± 650.0134.6 × 10−5M. jannaschiiProline210 ± 440.012 ± 0.0043120001Alanine2.0 ± 0.1431 ± 200.0635.3 × 10−6HumanProline1-cAs reported in Ref. 35.80 ± 200.18 ± 0.054401Alanine0.11 ± 0.0979 ± 100.000143.2 × 10−61-a kcat/K mis relative to proline in each set, which was set at 1.0. Individual kinetic parameters are based on the average of three determinations.1-b As reported in Ref. 26Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8920Crossref PubMed Scopus (121) Google Scholar.1-c As reported in Ref. 35Heacock D. Forsyth C.J. Shiba K. Musier-Forsyth K. Bioorganic Chem. 1996; 24: 273-289Crossref Scopus (108) Google Scholar. Open table in a new tab Cysteine was also activated quite well by all three enzymes, a phenomenon that has been observed in other systems (37Freist W. Sternbach H. Pardowitz I. Cramer F. J. Theor. Biol. 1998; 193: 19-38Crossref PubMed Scopus (23) Google Scholar). Based on initial rates of cysteine activation, we estimated that the ProRSs examined here activate cysteine with the following relativek cat/K m: 0.23 (E. coli), 0.026 (M. jannaschii), and 0.064 (human). These numbers are relative to proline activation by the respective ProRS. Thus, reductions in catalytic efficiency for cysteine activation by these three enzymes, assayed under the conditions described under "Experimental Procedures," range from only ∼4-fold for theE. coli enzyme to ∼38-fold for the M. jannaschii enzyme. That M. jannaschii ProRS can activate cysteine either in a tRNA-independent (38Lipman R.S.A. Sowers K.R. Hou Y.-M. Biochemistry. 2000; 39: 7792-7798Crossref PubMed Scopus (44) Google Scholar) or tRNA-dependent fashion (39Stathopoulos C. Li T. Longman R. Vothknecht U.C. Becker H.D. Ibba M. Söll D. Science. 2000; 287: 479-482Crossref PubMed Scopus (64) Google Scholar) has been previously noted. All kinetic parameters for amino acid activation reported here were determined in the absence of tRNA. Attempts to mischarge wild-type E. coli tRNAPro with alanine using E. coli ProRS were unsuccessful (Fig.1) (26Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8920Crossref PubMed Scopus (121) Google Scholar). In fact, we estimate that aminoacylation with alanine is at least 3.2 × 105-fold reduced compared with charging with cognate proline. This may be due to efficient pre- and post-transfer editing of alanine by E. coli ProRS (26Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8920Crossref PubMed Scopus (121) Google Scholar). Human ProRS is able to mischarge alanine onto tRNAPro detectably but at a level that is over 2.9 × 104-fold reduced compared with cognate charging. Interestingly, M. jannaschii ProRS mischarges tRNAPro with alanine at a rate that is only 6,800-fold reduced relative to charging with proline (Fig. 1). We also detected mischarging with cysteine of a tRNAPro transcript with M. jannaschii ProRS (TableII). The latter activity is examined in more detail in a separate report. 2R. S. A. Lipman, J. Wang, and Y.-M. Hou, submitted for publication. Table IISummary of Editing-Related Activities of E. coli, M. jannaschii, and Human ProRSProRS speciesAmino acidMisactivation (relativek cat/K m)Mischarging of tRNAProPretransfer editingPost-transfer editingE. coliAlanine+/−−++Cysteine+ND−NDM. jannaschiiAlanine+/−+++/−2-aThis activity is characterized as "weak" at the temperature at which the experiment was performed (37 °C). Because of technical reasons explained in the text, deacylation assays could not be carried out at the optimal temperature for M. jannaschii ProRS (≥60 °C).Cysteine++2-bAs also reported in footnote 2.+/−−2-bAs also reported in footnote 2.HumanAlanine+/−+/−−−Cysteine+ND+/−NDSymbols represent active (+), weak activity (+/−), and inactive (−). Weak activity is defined as >10-fold reduced relative to active (+) in each column. ND is "not determined".2-a This activity is characterized as "weak" at the temperature at which the experiment was performed (37 °C). Because of technical reasons explained in the text, deacylation assays could not be carried out at the optimal temperature for M. jannaschii ProRS (≥60 °C).2-b As also reported in footnote 2. Open table in a new tab Symbols represent active (+), weak activity (+/−), and inactive (−). Weak activity is defined as >10-fold reduced relative to active (+) in each column. ND is "not determined". ATP hydrolysis is diagnostic of pretransfer hydrolytic editing (3Jakubowski H. Goldman E. Microbiol. Rev. 1992; 56: 412-429Crossref PubMed Google Scholar). Similar to the E. colienzyme (26Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8920Crossref PubMed Scopus (121) Google Scholar), M. jannaschii ProRS stimulates ATP hydrolysis activity with the noncognate amino acid alanine (Fig.2 A) but, as expected, not in the presence of proline. As we previously determined for the E. coli enzyme, M. jannaschii ProRS also stimulates ATP hydrolysis in the presence of the proline analogs cis- andtrans-4-hydroxyproline. Only a very low level of ATP hydrolysis is observed in the presence of the four-membered ring analog of proline, azetidine-4-carboxylic acid, similar to the result obtained with E. coli ProRS (Fig. 2 A). Thus, the M. jannaschii enzyme parallels the E. coli enzyme in its pretransfer editing activity. M. jannaschii ProRS has been reported to possess dual specificity, activating cysteine and aminoacylating tRNACys, in addition to its normal proline-specific activity (38Lipman R.S.A. Sowers K.R. Hou Y.-M. Biochemistry. 2000; 39: 7792-7798Crossref PubMed Scopus (44) Google Scholar, 39Stathopoulos C. Li T. Longman R. Vothknecht U.C. Becker H.D. Ibba M. Söll D. Science. 2000; 287: 479-482Crossref PubMed Scopus (64) Google Scholar). In accordance with this dual specificity and in agreement with a recent independent report,2 we do not detect significant pretransfer editing against cysteine by M. jannaschii ProRS (Fig. 2 A). Human ProRS was also tested for its ability to stimulate ATP hydrolysis in the presence of noncognate amino acids. With the exception of very weak activity in the presence of cysteine, no significant stimulation was observed with any of the amino acids tested (Fig. 2 B). In particular, in contrast to the results obtained with the E. coli and M. jannaschii ProRSs, alanine is not edited by the human enzyme. As we had previously reported for E. coli ProRS (26Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8920Crossref PubMed Scopus (121) Google Scholar), A
Referência(s)