Lysyl-tRNA Synthetase-generated Lysyl-Adenylate Is a Substrate for Histidine Triad Nucleotide Binding Proteins
2006; Elsevier BV; Volume: 282; Issue: 7 Linguagem: Inglês
10.1074/jbc.m610530200
ISSN1083-351X
AutoresTsui-Fen Chou, Carston R. Wagner,
Tópico(s)RNA and protein synthesis mechanisms
ResumoHistidine triad nucleotide binding proteins (Hints) are the most ancient members of the histidine triad protein superfamily of nucleotidyltransferases and hydrolyases. Protein-protein interaction studies have found that complexes of the transcription factors MITF or USF2 and lysyl-tRNA synthetase (LysRS) are associated with human Hint1. Therefore, we hypothesized that lysyl-AMP or the LysRS·lysyl-AMP may be a native substrate for Hints. To explore the biochemical relationship between Hint1 and LysRS, a series of catalytic radiolabeling, mutagenesis, and kinetic experiments was conducted with purified LysRSs and Hints from human and Escherichia coli. After incubation of the E. coli or human LysRS with Hints and [α-32P]ATP, but not [α-32P]GTP, 32P-labeled Hints were observed. By varying time and the concentrations of lysine, Mg2+, or LysRS, the adenylation of Hint was found to be dependent on the formation of lysyl-AMP. Site-directed mutagenesis studies of the active site histidine triad revealed that Hint labeling could be abolished by substitution of either His-101 of E. coli hinT or His-112 of human Hint1 by either alanine or glycine. Ap4A, believed to be synthesized by LysRS in vivo, and Zn2+ were shown to inhibit the formation of Hint-AMP with an IC50 value in the low micromolar range. Consistent with pyrophosphate being an inhibitor for aminoacyl-tRNA synthetase, incubations in the presence of pyrophosphatase resulted in enhanced formation of Hint-AMP. These results demonstrate that the lysyl-AMP intermediate formed by LysRS is a natural substrate for Hints and suggests a potential highly conserved regulatory role for Hints on LysRS and possibly other aminoacyl-tRNA synthetases. Histidine triad nucleotide binding proteins (Hints) are the most ancient members of the histidine triad protein superfamily of nucleotidyltransferases and hydrolyases. Protein-protein interaction studies have found that complexes of the transcription factors MITF or USF2 and lysyl-tRNA synthetase (LysRS) are associated with human Hint1. Therefore, we hypothesized that lysyl-AMP or the LysRS·lysyl-AMP may be a native substrate for Hints. To explore the biochemical relationship between Hint1 and LysRS, a series of catalytic radiolabeling, mutagenesis, and kinetic experiments was conducted with purified LysRSs and Hints from human and Escherichia coli. After incubation of the E. coli or human LysRS with Hints and [α-32P]ATP, but not [α-32P]GTP, 32P-labeled Hints were observed. By varying time and the concentrations of lysine, Mg2+, or LysRS, the adenylation of Hint was found to be dependent on the formation of lysyl-AMP. Site-directed mutagenesis studies of the active site histidine triad revealed that Hint labeling could be abolished by substitution of either His-101 of E. coli hinT or His-112 of human Hint1 by either alanine or glycine. Ap4A, believed to be synthesized by LysRS in vivo, and Zn2+ were shown to inhibit the formation of Hint-AMP with an IC50 value in the low micromolar range. Consistent with pyrophosphate being an inhibitor for aminoacyl-tRNA synthetase, incubations in the presence of pyrophosphatase resulted in enhanced formation of Hint-AMP. These results demonstrate that the lysyl-AMP intermediate formed by LysRS is a natural substrate for Hints and suggests a potential highly conserved regulatory role for Hints on LysRS and possibly other aminoacyl-tRNA synthetases. Histidine triad nucleotide binding protein (Hint) 2The abbreviations used are: Hint1, histidine triad nucleotide binding protein1; hHint1, human Hint1; AMPCP, adenosine 5′-(α,β-methylene)diphosphate; HIT, histidine triad; Fhit, fragile histidine triad; Ap3A, diadenosine P1,P3-triphosphate; LysRS, lysyl-tRNA synthetase; hLysRS, human LysRS; ecLysU, E. coli LysRS.2The abbreviations used are: Hint1, histidine triad nucleotide binding protein1; hHint1, human Hint1; AMPCP, adenosine 5′-(α,β-methylene)diphosphate; HIT, histidine triad; Fhit, fragile histidine triad; Ap3A, diadenosine P1,P3-triphosphate; LysRS, lysyl-tRNA synthetase; hLysRS, human LysRS; ecLysU, E. coli LysRS. belongs to a histidine triad (HIT) superfamily that has a characteristic C-terminal active site motif, HXHXHXX, where X is a hydrophobic residue (1.Brenner C. Biochemistry. 2002; 41: 9003-9014Crossref PubMed Scopus (243) Google Scholar). HIT enzymes are a ubiquitous superfamily consisting primarily of nucleoside phosphoramidases, dinucleotide hydrolyases, and nucleotidyltransferases (1.Brenner C. Biochemistry. 2002; 41: 9003-9014Crossref PubMed Scopus (243) Google Scholar). Five distinct branches of HIT superfamily have been recently classified by a phylogenetic study of HIT proteins (2.Kijas A.W. Harris J.L. Harris J.M. Lavin M.F. J. Biol. Chem. 2006; 81: 13939-13948Abstract Full Text Full Text PDF Scopus (63) Google Scholar, 3.Ahel I. Rass U. El-Khamisy S.F. Katyal S. Clements P.M. McKinnon P.J. Caldecott K.W. West S.C. Nature. 2006; 443: 713-716Crossref PubMed Scopus (291) Google Scholar). Recently, Hint1 homologs isolated from rabbit (4.Bieganowski P. Garrison P.N. Hodawadekar S.C. Faye G. Barnes L.D. Brenner C. J. Biol. Chem. 2002; 277: 10852-10860Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), human (5.Chou T.-F. Bieganowski P. Shilinski K. Cheng J. Brenner C. Wagner C.R. J. Biol. Chem. 2005; 280: 15356-15361Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), chicken (6.Parks K.P. Seidle H. Wright N. Sperry J.B. Bieganowski P. Howitz K. Wright D.L. Brenner C. Physiol. Genomics. 2004; 20: 12-14Crossref PubMed Scopus (23) Google Scholar), yeast (4.Bieganowski P. Garrison P.N. Hodawadekar S.C. Faye G. Barnes L.D. Brenner C. J. Biol. Chem. 2002; 277: 10852-10860Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and Escherichia coli (5.Chou T.-F. Bieganowski P. Shilinski K. Cheng J. Brenner C. Wagner C.R. J. Biol. Chem. 2005; 280: 15356-15361Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) have been shown to be purine nucleoside phosphoramidases. Human Hint1 has been shown to associate with, and possibly regulate several transcription factors such as TFIIH (7.Korsisaari N. Makela T.P. J. Biol. Chem. 2000; 275: 34837-34840Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), MITF (8.Razin E. Zhang Z.C. Nechushtan H. Frenkel S. Lee Y.N. Arudchandran R. Rivera J. J. Biol. Chem. 1999; 274: 34272-34276Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 9.Lee Y.N. Nechushtan H. Figov N. Razin E. Immunity. 2004; 20: 145-151Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), and USF2 (10.Lee Y.N. Razin E. Mol. Cell. Biol. 2005; 25: 8904-8912Crossref PubMed Scopus (58) Google Scholar). Additionally, Hint1 knock out mice have been shown to have an increased susceptibility to the induction of ovarian and mammary tumors by the carcinogen dimethylbenzanthracene and to spontaneous tumors (11.Li H. Zhang Y. Su T. Santella R.M. Weinstein I.B. Oncogene. 2006; 25: 713-721Crossref PubMed Scopus (95) Google Scholar). Up-regulation of Hint1 and the significantly reduced in vivo tumorigenicity of 5-aza-dC-treated non-small-cell lung cancer cell line NCI-H522 suggested that hHint1 might be a tumor suppressor (12.Yuan B.Z. Jefferson A.M. Popescu N.C. Reynolds S.H. Neoplasia. 2004; 6: 412-429Crossref PubMed Scopus (47) Google Scholar). Recently, Weiske and Huber (13.Weiske J. Huber O. J. Biol. Chem. 2006; 281: 27356-27366Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) reported that Hint1 triggers apoptosis independent of its phosphoramidase activity (13.Weiske J. Huber O. J. Biol. Chem. 2006; 281: 27356-27366Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Human Hint2, which is 61% identical to Hint1, has recently been shown to be a mitochondrial apoptotic sensitizer that is down-regulated in hepatocellular carcinomas (14.Martin J. Magnino F. Schmidt K. Piguet A.-C. Lee J.-S. Semela D. St-Pierre M.V. Ziemiecki A. Cassio D. Mochly-Rosen D. Brenner C. Thorgeirsson S.S. Dufour J.-F. Gastroenterology. 2006; 130: 2179-2188Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Although Hints are efficient hydrolases of purine nucleoside phosphoramidates, cellular function and biochemical relevance of the enzymatic phosphoramidase activity has not been determined. The Fhit (fragile histidine triad) branch of the HIT superfamily is only found in eukaryotes. Like Hint, Fhit is also a homodimer with tumor suppressor activity (15.Ohta M. Inoue H. Cotticelli M.G. Kastury K. Baffa R. Palazzo J. Siprashvili Z. Mori M. McCue P. Druck T. Croce C.M. Huebner K. Cell. 1996; 84: 587-597Abstract Full Text Full Text PDF PubMed Scopus (1044) Google Scholar). Human Fhit is a diadenosine P1,P3-triphosphate (Ap3A) hydrolyase as well as phosphoramidase (16.Huang K. Arabshahi A. Wei Y. Frey P.A. Biochemistry. 2004; 43: 7637-7642Crossref PubMed Scopus (22) Google Scholar, 17.Huang K. Arabshahi A. Frey P.A. Eur. J. Org. Chem. 2005; 2005: 5198-5206Crossref Scopus (7) Google Scholar). The precise mechanism of action by which it affects tumor development is not well understood, although site-directed mutagenesis studies have indicated that the Fhit tumor suppressor function is likely not dependent on its Ap3A hydrolase activity (18.Pace H.C. Garrison P.N. Robinson A.K. Barnes L.D. Draganescu A. Rosler A. Blackburn G.M. Siprashvili Z. Croce C.M. Huebner K. Brenner C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5484-5489Crossref PubMed Scopus (133) Google Scholar). In contrast to the first two branches, the galactose-1-phosphate uridylyltransferase branch has been shown to be the second enzyme in the Leloir pathway necessary for galactose utilization (19.Frey P.A. Wong L.J. Sheu K.F. Yang S.L. Methods Enzymol. 1982; 87: 20-36Crossref PubMed Scopus (32) Google Scholar, 20.Frey P.A. FASEB J. 1996; 10: 461-470Crossref PubMed Scopus (376) Google Scholar). Although, galactose-1-phosphate uridylyltransferase is a homodimer with little overall sequence homology with Hint1 or Fhit, it does share some tertiary structure similarity (21.Brenner C. Garrison P. Gilmour J. Peisach D. Ringe D. Petsko G.A. Lowenstein J.M. Nat. Struct. Biol. 1997; 4: 231-238Crossref PubMed Scopus (127) Google Scholar). The fourth family is aprataxin, which is mutated in ataxia-oculomotor apraxia1 (22.Moreira M.C. Barbot C. Tachi N. Kozuka N. Uchida E. Gibson T. Mendonca P. Costa M. Barros J. Yanagisawa T. Watanabe M. Ikeda Y. Aoki M. Nagata T. Coutinho P. Sequeiros J. Koenig M. Nat. Genet. 2001; 29: 189-193Crossref PubMed Scopus (376) Google Scholar, 23.Date H. Onodera O. Tanaka H. Iwabuchi K. Uekawa K. Igarashi S. Koike R. Hiroi T. Yuasa T. Awaya Y. Sakai T. Takahashi T. Nagatomo H. Sekijima Y. Kawachi I. Takiyama Y. Nishizawa M. Fukuhara N. Saito K. Sugano S. Tsuji S. Nat. Genet. 2001; 29: 184-188Crossref PubMed Scopus (333) Google Scholar) and possesses phosphoramidase and Ap4A hydrolase activity as well as DNA/RNA binding properties (2.Kijas A.W. Harris J.L. Harris J.M. Lavin M.F. J. Biol. Chem. 2006; 81: 13939-13948Abstract Full Text Full Text PDF Scopus (63) Google Scholar); recently, the physiological substrate for aprataxin has been shown to be abortive adenylated DNA ligation intermediates (3.Ahel I. Rass U. El-Khamisy S.F. Katyal S. Clements P.M. McKinnon P.J. Caldecott K.W. West S.C. Nature. 2006; 443: 713-716Crossref PubMed Scopus (291) Google Scholar). The last family is the scavenger mRNA decapping enzyme, DcpS/DCS-1, a 7-methyl-GpppG hydrolase (24.Liu H. Rodgers N.D. Jiao X. Kiledjian M. EMBO J. 2002; 21: 4699-4708Crossref PubMed Scopus (203) Google Scholar, 25.Kwasnicka D.A. Krakowiak A. Thacker C. Brenner C. Vincent S.R. J. Biol. Chem. 2003; 278: 39051-39058Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Recently, hHint1 has been isolated from complexes with lysyl-tRNA synthetase (LysRS) and MITF or USF2 transcription factors (9.Lee Y.N. Nechushtan H. Figov N. Razin E. Immunity. 2004; 20: 145-151Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 10.Lee Y.N. Razin E. Mol. Cell. Biol. 2005; 25: 8904-8912Crossref PubMed Scopus (58) Google Scholar). To elucidate the biochemical connection between LysRS and Hint1, both E. coli LysRS (ecLysU) and human LysRS (hLysRS) were purified and shown to label echinT and hHint1 with [α-32P]ATP but not with [γ-32P]ATP, [α-32P]GTP, or [γ-32P]GTP. This was found to be consistent with chemical degradation and mutagenic experiments revealing that the Hint-AMP intermediate formed upon interaction with LysRS contained a phosphoramidate linkage between AMP and a strictly conserved and catalytically essential active site histidine (His-112 of hHint1, Fig. 1). Furthermore, by varying individual components in the aminoacylation reaction, the formation of the Hint-AMP intermediate was found to be dependent on the formation of the lysyl-AMP intermediate. Hence, Hints may function in part to regulate the catalytic activity of LysRS by providing a possible new mechanism of pre-transfer editing or by scavenging highly reactive inappropriately released aminoacyl-adenylates. Moreover, the interaction of Hint1 with transcription factors such as USF2 and MITF may be mediated by the adenylation of Hint1 by LysRS. Protein Purification−The E. coli BS68 strain harboring the pBAS39 (derived from pET3a) plasmid for expressing ecLysU as a C-terminal His6-tag fusion protein was a gift from Dr. Paul Schimmel (The Scripps Research Institute) (26.Steer B.A. Schimmel P. Biochemistry. 1999; 38: 4965-4971Crossref PubMed Scopus (13) Google Scholar). The plasmid pM368 encoding hLysRS expresses a fusion protein containing the N-terminal MRGSHHHHHHSSGWVD sequence appended to full-length hLysRS was a gift from Dr. Karin Musier-Forsyth (University of Minnesota) (27.Shiba K. Stello T. Motegi H. Noda T. Musier-Forsyth K. Schimmel P. J. Biol. Chem. 1997; 272: 22809-22816Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Both LysRS enzymes were purified by using Ni2+-agarose binding according to a previously published procedure (27.Shiba K. Stello T. Motegi H. Noda T. Musier-Forsyth K. Schimmel P. J. Biol. Chem. 1997; 272: 22809-22816Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Wild type and mutants of hHint1 and echinT were purified by an AMP-agarose column from echinT knock out strain (BB2) as described previously (5.Chou T.-F. Bieganowski P. Shilinski K. Cheng J. Brenner C. Wagner C.R. J. Biol. Chem. 2005; 280: 15356-15361Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Labeling of Hints by Purified E. coli LysU−E. coli LysU (6.25 μm) was incubated with [α-32P]ATP or [α-32P]GTP (0.33 μm, 800 Ci/mmol, MP Biomedicals) in buffer A (10 μl, 25 mm Tris HCl, pH 7.8, 100 mm NaCl, 2 mm MgCl2, 1 mm dithiothreitol, protease inhibitor tablet (Roche Applied Science)) at 23 °C for 10 min followed by the addition of either buffer A (5 μl), echinT (5 μl, 1.25, 3, or 7.5 μm), hHint1 (5 μl, 1.25, 3, or 7.5 μm), or the chimera mutant (5 μl, 1.25, 3, or 7.5 μm) and incubated for 10 min. The reaction was terminated by the addition of SDS sample buffer (4×,5 μl, Invitrogen). The reaction mixture was boiled for 10 min, and the proteins were separated by SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane. Labeled proteins were visualized by subjecting dried polyvinylidene difluoride membranes to autoradiography with a storage phosphor screen for 12 h followed by scanning with a Storm 840 PhosphorImager. Lysyl-AMP-dependent Adenylation of Hints by E. coli LysU and Human LysRS−The concentration of ecLysU, hLysRS, lysine, [α-32P]ATP, Mg 2+, Zn 2+, nucleotides, yeast inorganic pyrophosphatase (Sigma), Hint proteins, and incubation time period for each experiment is described in the legends to Figs. 3, 4, 5, 6, 7, 8, 9. The reactions were terminated by the addition of SDS sample buffer (4×), and samples were analyzed as described above. Quantitation of the intensity of the 32P signal was carried out with ImageQuant software (GE Healthcare).FIGURE 4Time dependence of Hint adenylation by ecLysU. Hint-AMP formation was assayedat23 °Coveran8-mintimecourseafterincubationofecLysU (0.022 μm) in buffer A containing lysine (2.2 μm) with [α-32P]ATP (0.35 μm) for 1 min. After the addition of echinT (1.4 μm), aliquots were withdrawn at 1-min intervals and quenched with SDS sample buffer. The steady-state level of Hint-AMP was maintained for 2 min, then the intensity was decreased from 3 to 8 min.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Adenylation of ecLysU by echinT. Incubation of ecLysU (0.022 μm) was carried out at 23 °C in buffer A containing lysine (2.2 μm) with [α-32P]ATP (0.35 μm) for 1 min followed by the addition of echinT (0.056–14 μm) and incubation for 1 min. Maximal Hint-AMP formation was observed at 2.8 μm echinT. Formation of ecLysU-AMP was observed after the concentration of echinT reached 1.4 μm. The intensity of the ecLysU-AMP was increased with higher concentrations of echinT (2.8 ∼ 4 μm). Moreover, the ecLysU/echinT-AMP complex or multiple adenylated ecLysU was observed at 14 μm echinT.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Adenylation of echinT is dependent on the concentration of lysine and Mg2+. Incubation of ecLysU (0.025 μm) was carried out at 23 °C in buffer A containing lysine (0 ∼ 2500 μm) with [α-32P]ATP (0.32 μm) for 1 min followed by the addition of echinT (2.5 μm) for an additional 1 min (A) and in buffer A containing lysine (2.5 μm) and Mg2+ (0 ∼ 3000 μm) with [α-32P]ATP (0.32 μm) for 1 min followed by the addition of echinT (2.5 μm) for 1 min (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Inhibition of Hint-AMP formation by Zn2+ and Ap4A. Incubation of ecLysU (0.025 μm) was carried out at 23 °C in buffer A containing lysine (2.5 μm) with [α-32P]ATP (0.32 μm) with various concentrations of Zn2+ (0 ∼ 1500 μm) for 1 min followed by the addition of echinT (2.5 μm) for 1 min (A) and various concentrations of Ap4A(0 ∼ 1500 μm) for 1 min followed by the addition of echinT (2.5 μm) for 1 min (B). The IC50 of Ap4A on Hint AMP formation was about 15 μm. In addition, Ap3A and ATP exhibited similar inhibitory effects, whereas GTP displayed less effect. AMP and GMP have no-observable effect.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 8Dependence of Hint adenylation on the concentration of echinT and ecLysU. Adenylation was carried out in buffer A containing lysine (2.5 μm) with [α-32P]ATP (0.32 μm) at 23 °C. A, incubations with ecLysU (0.025 μm) for 1 min followed by the addition of echinT (0.05–12.5 μm) for 1 min. Steady-state levels of Hint-AMP were observed when the concentration of echinT reached 2.5 μm. B, incubations with ecLysU (0.025 ∼ 14 μm) for 1 min followed by the addition of echinT (6.25 μm) for 1 min. The intensity of Hint-AMP is proportional to the increased concentration of ecLysU.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 9Adenylation of Hints by ecLysU and hLysRS. Labeling was carried out in buffer A containing lysine (35 μm) and inorganic pyrophosphatase (0.02 unit/μl) with [α-32P]ATP (0.9 μm) at 23 °C for 1 min followed by the addition of Hint proteins (3.6 μm) for 1 min with ecLysU (0.1 μm)(A) and hLysRS (1 μm)(B). WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Measurement of AMP Production by TLC Assay−The TLC assay was employed to measure the formation of AMP by aminoacyl-tRNA synthetase (28.Bullock T.L. Uter N. Nissan T.A. Perona J.J. J. Mol. Biol. 2003; 328: 395-408Crossref PubMed Scopus (80) Google Scholar, 29.Hati S. Ziervogel B. Sternjohn J. Wong F.C. Nagan M.C. Rosen A.E. Siliciano P.G. Chihade J.W. Musier-Forsyth K. J. Biol. Chem. 2006; 281: 27862-27872Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). E. coli LysU (1 μm) was added to the reaction mixture containing ATP (250 μm), lysine (200 μm), [α-32P]ATP (7.5 μm) in buffer B (100 mm Tris HCl, pH 7.0, 10 mm NaCl, 10 mm MgCl2, 5 mm dithiothreitol, 0.03 unit/μl inorganic pyrophosphatase) at 23 °C. Aliquots (2 μl) were with-drawn and quenched in ice-cold sodium acetate solution (200 mm, pH 5.0). After 10 or 30 min, echinT (2.5 μm) was added to the remaining reaction mixture, and AMP formation was monitored for an additional 10 min. Aliquots of quenched reactions (1 μl) were spotted in duplicate on polyethyleneimine-cellulose plates (20 × 10 cm, Selecto Scientific) pre-developed with water. The air-dried TLC plate was developed in ammonium acetate (100 mm), 5% acetic acid solution. The 32P signal was detected and quantitated as described above. The Chimeric Mutant of hHint1 and echinT−Human Hint1 and echinT shared 50% sequence identity over 107 alignable amino acids, but their C termini are distinct. The C terminus may play an important role on determining substrate specificity. Homology modeling studies revealed that the C termini of echinT are likely to reside in a similar location to that of the hHint1. 3T.-F. Chou, Y. Y. Sham, and C. R. Wagner, unpublished data. To address this possibility, a human/E. coli chimera was created by replacing the C terminus of hHint1 (Gln-120 to Gly-126) with the C terminus of echinT (Pro-109 to Leu-119). Nucleotidylation of Hints by ecLysU−Adenylation but not guanylation of Hints was observed after incubation with ecLysU (Fig. 2A), whereas the control experiments without ecLysU (Fig. 2A, lanes 1, 5, and 8) revealed negligible labeling of Hints. Adenylation of echinT (Fig. 2A, lanes 3 and 4, 13 kDa), hHint1 (Fig. 2A, lanes 6 and 7, 14 kDa), and the chimera mutant (Fig. 2A: lanes 9 and 10, 14 kDa) was compared which demonstrate that labeling of echinT and the chimera was more intense than the labeling of hHint1, indicating the potential role of the C-terminal loop in facilitating labeling. In contrast to adenylation, incubation of ecLysU with [α-32P]-GTP revealed weak labeling of ecLysU (59 kDa) after the addition of hHint1 (Fig. 2B, lane 3) and the chimera (Fig. 2B, lane 4) but no labeling with only buffer A (Fig. 2B, lane 1) or echinT (Fig. 2B, lane 2). Enhanced Adenylation of Hint by ecLysU by the Addition of Lysine−To first investigate whether the lysyl-tRNA synthetase activity associated with ecLysU was responsible for the adenylation of Hints, the effect of lysine on Hint-AMP formation was evaluated. Incubation of ecLysU (0.22 μm) was carried out at 23 °C for 10 min with [α-32P]ATP (0.38 μm) in buffer A followed by the addition of echinT (0.28 or 2.8 μm) for 10 min (Fig. 3, lanes 2 and 4) and in buffer A containing lysine (2.2 μm) followed by the addition of echinT (0.28 or 2.8 μm) for 10 min (lanes 3 and 5). The addition of lysine (2.2μm) enhanced Hint-AMP formation by 11-fold, when the ecLysU (0.22 μm) and echinT (0.28 μm) were comparable in concentration. Consistent with a saturable process, when the amount of echinT was increased by 10-fold, only a 5-fold increase in labeling was observed. Because the small amount of Hint-AMP was observed in the absence of added lysine (Fig. 3, lanes 2 and 4), it is highly likely that a small amount of contaminating lysine or lysyl-AMP bound with ecLysU is present with the purified enzyme. In addition, at the higher echinT concentration, we do observe small amounts of labeled ecLysU (Fig. 3, lanes 4 and 5, 59 kDa), possibly indicating that at high enough concentrations of Hint, adenylation of ecLysU can be observed. Time Dependence of Hint Adenylation by ecLysU−To evaluate the rate of adenylate-Hint formation, after preincubation of ecLysU (0.022 μm), lysine (2.2 μm), and [α-32P]ATP (0.38 μm) for 1 min, excess echinT (1.4 μm) was added to initiate the reaction; the reaction was carried out for an additional 8 min with aliquots removed and quenched at 1-min intervals. As shown in Fig. 4, maximal Hint-AMP formation was observed between 1 and 3 min after initiation. Consistent with rapid consumption of ATP by ecLysU and turnover of the Hint-AMP intermediate, over the course of the next 5 min, a 6-fold decrease in the intensity of echinT labeling was observed. In subsequent experiments, the labeling of echinT was carried out for maximum of 1 min. Adenylation of ecLysU by echinT−Incubation of ecLysU (0.022 μm) with lysine and [α-32P]-ATP for 1 min followed by the addition of various concentrations of echinT (0.056–14 μm) revealed that the intensity of the Hint-AMP was proportional to the added amount of echinT, reaching a plateau between 1.4 and 2.8 μm. Moreover, labeling of ecLysU was noticeable when incubations were carried out with at least a 56-fold excess of echinT. The prevalence of an additional higher Mr band, consistent in size with covalently linked ecLysU and echinT, was observed at the highest echinT concentration (14 μm) (Fig. 5). The possibility that this band may result from the labeling of LysRS at multiple sites cannot be ruled out. Labeling of the high Mr band and ecLysU is likely to suppress the amount of observed echinT-AMP. Adenylation of Hint Is Dependent on the Concentration of Lysine and Mg2+−Because lysine is one of the substrates and Mg2+ is absolutely required for lysyl-AMP formation (30.Desogus G. Todone F. Brick P. Onesti S. Biochemistry. 2000; 39: 8418-8425Crossref PubMed Scopus (75) Google Scholar), it is of interest to determine the effect of both molecules on adenylation of echinT in more detail. Hence, various concentrations of lysine (0.025–2500 μm) were examined to reveal a saturation-type behavior with respect to the amount of lysine; under these conditions maximal intensity was observed at 25 μm lysine (Fig. 6A). In addition, labeling was observed only when Mg2+ was present in the reaction mixture, with maximal intensity reached at a concentration of 1500 μm (Fig. 6B). Taken together, the observed reliance of echinT adenylation on lysine and Mg2+ is consistent with a labeling reaction dependent on the formation of lysyl-AMP by ecLysU. Inhibition of Hint Adenylation by Zn2+,Ap4A, and Ap3A− Divalent Zn2+ has been shown to stimulate Ap4A formation by ecLysU. The mechanism has been shown to rely on the ability of Zn2+ to stimulate nucleophilic attack by the γ-phosphate of ATP on lysyl-AMP (31.Theoclitou M.E. Wittung E.L. Hindley A.D. El-Thaher T.S.H. Miller A.D. J. Chem. Soc. Perkin Trans. I. 1996; 16: 123-125Google Scholar). In addition, Zn2+, but not Ap4A, has been shown to inhibit Hint activity with an IC50 of (9.5 ± 2.1) μm. 4T.-F. Chou and C. R. Wagner, unpublished data. Therefore, we chose to test the effect of Zn2+, Ap4A, and other nucleotides on the formation of Hint-AMP. As shown in Fig. 7, Zn2+ (15 μm) inhibited Hint-AMP formation by 80%, and Ap4A inhibited Hint-AMP formation with an IC50 of 15 μm. Moreover, about 70–90% inhibition was observed for incubations with 150 μm Ap4A, Ap3A, or ATP. Consistent with the preference of LysRS for ATP and not GTP, incubations with 150 μm GTP decreased Hint-AMP by only 50%, whereas inhibition by either AMP or GMP was not observed (supplemental Fig. 1). These results provided additional evidence that lysyl-AMP is required for adenylation of echinT. Dependence of Hint Adenylation on the Concentration of echinT and ecLysU−To examine the concentration effect of echinT and ecLysU on Hint-AMP intermediate formation, we first fixed the ecLysU concentration (0.025 μm) and varied the concentration of echinT from 0.05 to 12.5 μm. A plateau in the amount of echinT labeling was observed over the concentration range 2.5–12.5 μm echinT (Fig. 8A). When the ecLysU concentration was varied from 0.025 to 14 μm with a fixed echinT concentration (6.25 μm), the intensity of Hint-AMP was found to increase with increasing amounts of ecLysU (Fig. 8B). Effect of Inorganic Pyrophosphate (PPi) on Hint-AMP Formation−Yeast inorganic pyrophosphatase, which hydrolyzes PPi, has been used to enhance the catalytic activity of LysRS by reducing product inhibition by PPi (32.Jakubowski H. Biochemistry. 1999; 38: 8088-8093Crossref PubMed Scopus (54) Google Scholar). The addition of inorganic pyrophosphatase to aminoacyl-tRNA synthetase reaction mixtures has been suggested to more closely mimic the in vivo conditions for aminoacylation (33.Khvorova A. Motorin Y. Wolfson A.D. Nucleic Acids Res. 1999; 27: 4451-4456Crossref PubMed Scopus (11) Google Scholar, 34.Wolfson A.D. Uhlenbeck O.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5965-5970Crossref PubMed Scopus (108) Google Scholar). Therefore, adenylation of echinT was also carried out with and without inorganic pyrophosphatase to evaluate the effect of PPi on Hint-AMP formation. As shown in supplemental Fig. 2, in the presence of inorganic pyrophosphatase-enhanced Hint-AMP formation was observed (3-fold). Effect of Hint on AMP Formation by ecLysU−To further establish th
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