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• W2089654647 abstract "When the genome of the thermophilic archaeonPyrococcus horikoshii was sequenced, a gene homologous to the mammalian gene for an acylamino acid-releasing enzyme (EC 3.4.19.1) was found in which the enzyme's proposed active residues were conserved. The P. horikoshii gene comprised an open reading frame of 1,896 base pairs with an ATG initiation codon and a TAG termination codon, encoding a 72,390-Da protein of 632 amino acid residues. This gene was overexpressed in Escherichia coliwith the pET vector system, and the resulting enzyme showed the anticipated amino-terminal sequence and high hydrolytic activity for acylpeptides. This enzyme was concluded to be the first acylamino acid-releasing enzyme from an organism other than a eukaryotic cell. The existence of the enzyme in archaea suggests that the mechanisms of protein degradation or initiation of protein synthesis or both in archaea may be similar to those in eukaryotes. The enzyme was stable at 90 °C, with its optimum temperature over 90 °C. The specific activity of the enzyme increased 7–14-fold with heat treatment, suggesting the modification of the enzyme's structure for optimal hydrolytic activity by heating. This enzyme is expected to be useful for the removal of Nα-acylated residues in short peptide sequence analysis at high temperatures. When the genome of the thermophilic archaeonPyrococcus horikoshii was sequenced, a gene homologous to the mammalian gene for an acylamino acid-releasing enzyme (EC 3.4.19.1) was found in which the enzyme's proposed active residues were conserved. The P. horikoshii gene comprised an open reading frame of 1,896 base pairs with an ATG initiation codon and a TAG termination codon, encoding a 72,390-Da protein of 632 amino acid residues. This gene was overexpressed in Escherichia coliwith the pET vector system, and the resulting enzyme showed the anticipated amino-terminal sequence and high hydrolytic activity for acylpeptides. This enzyme was concluded to be the first acylamino acid-releasing enzyme from an organism other than a eukaryotic cell. The existence of the enzyme in archaea suggests that the mechanisms of protein degradation or initiation of protein synthesis or both in archaea may be similar to those in eukaryotes. The enzyme was stable at 90 °C, with its optimum temperature over 90 °C. The specific activity of the enzyme increased 7–14-fold with heat treatment, suggesting the modification of the enzyme's structure for optimal hydrolytic activity by heating. This enzyme is expected to be useful for the removal of Nα-acylated residues in short peptide sequence analysis at high temperatures. The acylamino acid-releasing enzyme (AARE) 1The abbreviations used are: AARE, acylamino acid-releasing enzyme; Ac-, Nα-acetyl; f-, Nα-formyl; pNA,p-nitroanilide; α-MSH, α-melanocyte-stimulating hormone; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; DMF, N, N-dimethylformamide; DSC, differential scanning calorimetry; AAREP, AARE from P. horikoshii; HAAREP, heat-activated AAREP. catalyzes the NH2-terminal hydrolysis ofNα-acylpeptides to releaseNα-acylated amino acids (1Tsunasawa S. Narita K. Ogata K. J. Biochem. ( Tokyo ). 1975; 77: 89-102PubMed Google Scholar). AARE has been used for removal of Nα-acylated residues in protein sequence analysis. Until now, AARE has been isolated only from eukaryotic cells (1Tsunasawa S. Narita K. Ogata K. J. Biochem. ( Tokyo ). 1975; 77: 89-102PubMed Google Scholar, 2Gade W. Brown J.L. J. Biol. Chem. 1978; 253: 5012-5018Abstract Full Text PDF PubMed Google Scholar, 3Tsunasawa S. Imanaka T. Nakazawa T. J. Biochem. ( Tokyo ). 1983; 93: 1217-1220Crossref PubMed Scopus (11) Google Scholar, 4Marks N. Lo E.-S. Stern F. Danho W. J. Neurochem. 1983; 41: 201-208Crossref PubMed Scopus (14) Google Scholar) and classified as its own serine protease subfamily (5Rawling N.D. Polgar L. Barrett A.J. Biochem. J. 1991; 279: 907-908Crossref PubMed Scopus (129) Google Scholar, 6Polgar L. FEBS Lett. 1992; 311: 281-284Crossref PubMed Scopus (58) Google Scholar). The physiological role of the enzyme is not clear, although it has been suggested that it affects the processing or sorting of proteins (7Palmiter R.D. Gagnon J. Walsh K.A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 94-98Crossref PubMed Scopus (199) Google Scholar, 8Rubenstein P.A. Martin D.J. J. Biol. Chem. 1983; 258: 11354-11360Abstract Full Text PDF PubMed Google Scholar) in eukaryotic cells. From eukaryotic cells, some AARE genes have already been cloned (9Mita M. Asada K. Uchimura Y. Kimizuka F. Kato I. Sakiyama F. Tsunasawa S. J. Biochem. ( Tokyo ). 1989; 106: 548-551Crossref PubMed Scopus (46) Google Scholar, 10Kobasyashi K. Lin L.W. Yeadon J.E. Klickstein L.B. Smith J.A. J. Biol. Chem. 1989; 264: 8892-8899PubMed Google Scholar). However, the production and expression of AARE from these genes withinEscherichia coli have not been carried out. Pyrococcus horikoshii (OT3) is one of the thermophilic archaea collected from a volcanic vent in the Okinawa trough (11Swinbanks D. Nature News. 1995; 374: 583Crossref PubMed Scopus (4) Google Scholar). The optimum growth temperature of this archaeon ranges from 90 to 105 °C. Most of the proteins from P. horikoshii are thought to be thermostable and active at high temperature. The size of its genome is about 2 Mb, and the guanine-cytosine content is relatively low. At the National Institute of Technology and Evaluation (Tokyo, Japan), sequencing of this genome is in progress (11Swinbanks D. Nature News. 1995; 374: 583Crossref PubMed Scopus (4) Google Scholar). From the genome sequencing in P. horikoshii we found a gene that had some homology with a gene for AARE from pig liver (9Mita M. Asada K. Uchimura Y. Kimizuka F. Kato I. Sakiyama F. Tsunasawa S. J. Biochem. ( Tokyo ). 1989; 106: 548-551Crossref PubMed Scopus (46) Google Scholar, 12Miyagi M. Sakiyama F. Kato I. Tsunasawa S. J. Biochem. ( Tokyo ). 1995; 118: 771-779Crossref PubMed Scopus (20) Google Scholar). Therefore, we cloned the gene from P. horikoshii and attempted to express the enzyme in E. coli and examine the characteristics of the expressed enzyme. The host E. coli BL21(DE3) and the vector pET11a were obtained from Novagen (Madison, WI). ThePfu DNA polymerase, restriction enzymes, and ligation kit were purchased from Takara Shuzo (Otsu, Shiga, Japan). TheNα-acetylamino acid p-nitroanilide derivatives (Ac-amino acid-pNA) were purchased from Sigma (St. Louis, MO) and Bachem (Bubendorf, Switzerland). The acylpeptides Ac-Ala-Ala, Ac-Met-Ala, f-Met-Ala, f-Met-Ala-Ser, f-Met-Leu-Gly, and f-Ala-Ala-Ala were also from Bachem. The other acylpeptides Ac-Met-Ala-Ala-Ala-Ala-Ala, Ac-Ala-Ala-Ala-Ala, Ac-Ala-Ala-Ala-Ala-Ala-Ala, f-Met-Ala-Ala-Ala-Ala-Ala, and f-Ala-Ala-Ala-Ala-Ala-Ala were purchased from Peptide Institute Inc. (Minou, Osaka, Japan). α-Melanocyte-stimulating hormone (α-MSH) was purchased from Funakoshi (Tokyo, Japan). The synthesis of DNA primers and the sequencing of proteins were performed by the custom service center of Takara Shuzo. The DNA sequencing was carried out with ABI model 373 sequencer (Perkin-Elmer, Applied Biosystems Div., Foster City, CA). All other chemicals were of the highest reagent grade commercially available. The genome of P. horikoshii was sequenced by the method of Kaneko et al.(13Kaneko T. Tanaka A. Sato S. Kotani H. Sazuka T. Miyajima N. Sugiura M. Tabata S. DNA Res. 1995; 2: 153-166Crossref PubMed Scopus (263) Google Scholar). The gene that was homologous to the mammalian gene for AARE was found by BLAST search (14Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar). The gene was amplified by the polymerase chain reactionmethod using two primers with unique restriction sites. Amplification of the gene by polymerase chain reaction was carried out at 94 °C for 1 min, 55 °C for 2 min, and 72 °C for 3 min, for 35 cycles using Pfu DNA polymerase. The amplified gene was hydrolyzed by the restriction enzymes and inserted in pET11a cut by the same restriction enzymes. The amplified gene was expressed using the pET11a vector system in the host E. coli BL21(DE3) according to the manufacturer's instructions. The host E. coliBL21(DE3) was transformed by the constructed plasmid. The transformant cell was grown in 2YT medium (1% yeast extract, 1.6% tryptone, and 0.5% NaCl) containing ampicillin (100 μg/ml) at 37 °C. After incubation with shaking at 37 °C until theA600 reached 0.6–1.0, the induction was carried out by adding isopropyl β-d-thiogalactopyranoside at a final concentration of 1 mm and shaking for 4 h at 37 °C. The concentration of the enzyme was determined with Coomassie protein assay reagent (Pierce Chemical Company, Rockford, IL) using bovine serum albumin as the standard protein. After induction, the transformant cells were harvested by centrifugation and disrupted with oxide aluminum in 50 mm Tris-HCl buffer (pH 8.0) containing 0.6 m NaCl. After incubation with DNase I (from bovine pancreas; Sigma) for 30 min at room temperature, the crude extract was heated at 85 °C for 30 min. The supernatant obtained by centrifugation was dialyzed against 50 mm Tris-HCl buffer (pH 8.0). The dialyzed sample was loaded on a HiTrap Q column (Pharmacia, Uppsala, Sweden). The column was washed with 50 mm Tris-HCl buffer (pH 8.0) and eluted with a linear gradient (0–1.0 m NaCl in the same buffer). The fractions that showed protein of a similar molecular mass(70 kDa, SDS-PAGE) calculated from the amino acid sequence were concentrated by a Centricon 10 filter (Amicon Inc., Beverly, MA). The concentrated material was loaded on a HiLoad Superdex 200 column (Pharmacia) and eluted with 100 mm Tris-HCl buffer (pH 8.0) containing 1.0m NaCl. The fractions demonstrating only one protein band with a molecular mass of 70 kDa by SDS-PAGE were collected and used for the detailed characterizations of the enzyme. The molecular weight of the enzyme was determined by SDS-PAGE performed on a 4–15% gradient gel in the Phast System (Pharmacia). Protein bands were visualized by staining with Coomassie Brilliant Blue. The molecular weight was also determined by high performance liquid chromatography (HPLC) and light-scattering photometry. The HPLC was performed on a Superdex 200 column (Pharmacia), and the elution was carried out using 100 mm Tris-HCl buffer (pH 8.0) containing 1.0 m NaCl at 1.5 ml/min at room temperature. The eluted protein was detected by its absorbance at 280 nm. The light-scattering photometer was conducted at room temperature with a DLS-700S light-scattering photometry (Otsuka Denshi, Shiga, Japan) calibrated with benzene (15Pike E.R. Pomeroy W.R.M. Vaughan J.M. J. Chem. Phys. 1975; 62: 3188-3192Crossref Scopus (160) Google Scholar) at 633 nm and analyzed by the method of Kamata and Nakahara (16Kamata T. Nakahara H. J. Colloid Int. Sci. 1973; 43: 89-96Crossref Scopus (32) Google Scholar). Optical clarification was performed with polyvinylidene fluoride filters. The specific refractive index increment (dn/dc) was obtained with a KMX-16 refractometer (Chromatix Inc., Sunnyvale, CA at the same wavelength, calibrated with NaCl solution. The activity of the enzyme was determined using Ac-amino acid-pNA and acylpeptides. The enzyme was incubated at 85 °C with the substrates in 50 mm sodium acetate buffer (pH 5.4) containing 0.6 m NaCl and 5%N, N-dimethylformamide (DMF), and the released products were measured. The activity toward the Ac-amino acid-pNAs was calculated using the absorption coefficient ε406 = 9.91 mm−1 ofpNA released (17Kobayashi K. Smith J.A. J. Biol. Chem. 1987; 262: 11435-11445Abstract Full Text PDF PubMed Google Scholar). The activity toward the acylpeptides was measured by the detection of the exposed α-NH2 group with the cadmium-ninhydrin colorimetric method (18Doi E. Shibata D. Matoba T. Anal. Biochem. 1981; 118: 173-184Crossref PubMed Scopus (420) Google Scholar). The analysis of the products from the peptides was performed by HPLC on an ODS-80Ts column (4.6-mm inner diameter × 25 cm) containing TSK gel (Tosoh, Tokyo, Japan). The flow rate was 0.7 ml/min with 95% water, 5% acetonitrile, and 0.1% trifluoroacetic acid. The activity toward α-MSH was examined by a PSQ-1 protein sequencer (Shimazu, Kyoto, Japan) at the custom service center of Takara Shuzo. 1 unit of activity corresponds to the amount of enzyme which catalyzes the hydrolysis of 1 μmol of substrate/min. Thermostability of the enzyme was measured by the circular dichroism (CD) and the differential scanning calorimetry (DSC). CD was measured with a CD spectrometer (model 62A DS) (Aviv Instrument, Lakewood, NJ) utilizing a 5.0-mm path length quartz cell in the far UV region. The scan rate of the temperature was 1 K/min. The measurement was carried out in 50 mm sodium acetate buffer (pH 5.4) containing 0.6 m NaCl. The experiments of DSC were performed in a model DSC5100 calorimeter (Calorimetry Sciences Corp., Provo, UT). A scan rate of 1 K/min was used throughout. Before measurement, the sample was dialyzed against 50 mm sodium acetate buffer (pH 5.4) containing 0.6m NaCl and degassed with an aspirator for 15 min. Instrument base lines were established with both cells filled with dialysate; the reference cell remained filled with dialysate during the protein scans. In the genome sequenced fromP. horikoshii, we found a gene that contained 1,896 base pairs and showed about 20% identity with the AARE gene from pig liver (Fig. 1) (12Miyagi M. Sakiyama F. Kato I. Tsunasawa S. J. Biochem. ( Tokyo ). 1995; 118: 771-779Crossref PubMed Scopus (20) Google Scholar). The open reading frame was preceded by AT-rich regions in which a putative ribosome binding site GGTGAT at position −4 and a putative promoter consensus TTATAT at position −33 from ATG initiation site were found. This consensus resembles the eukaryotic TATA box and has been confirmed to be the archaeal consensus sequence TT(A/T)(T/A)AX, as determined by analysis of more than 80 archaeal promoters (19Palmer J.R. Daniels C.J. J. Bacteriol. 1995; 177: 1844-1849Crossref PubMed Scopus (100) Google Scholar). The protein encoded consists of 632 amino acids, making it smaller than AARE (732 amino acids) from a mammal. However, the proposed active residues (Ser, Asp, and His) of AARE, called “catalytic triad residues” (5Rawling N.D. Polgar L. Barrett A.J. Biochem. J. 1991; 279: 907-908Crossref PubMed Scopus (129) Google Scholar, 6Polgar L. FEBS Lett. 1992; 311: 281-284Crossref PubMed Scopus (58) Google Scholar, 12Miyagi M. Sakiyama F. Kato I. Tsunasawa S. J. Biochem. ( Tokyo ). 1995; 118: 771-779Crossref PubMed Scopus (20) Google Scholar), were conserved (Fig. 1). Furthermore, the sequence homology in the Ser, Asp, and His regions (6Polgar L. FEBS Lett. 1992; 311: 281-284Crossref PubMed Scopus (58) Google Scholar) of a new family of serine-type peptidases (5Rawling N.D. Polgar L. Barrett A.J. Biochem. J. 1991; 279: 907-908Crossref PubMed Scopus (129) Google Scholar) was also observed in this protein. The residues Tyr-492 in the Ser region and Glu-602 in the His region of this protein are not conserved in AARE, but dipeptidyl peptidase (6Polgar L. FEBS Lett. 1992; 311: 281-284Crossref PubMed Scopus (58) Google Scholar). These results suggest that this protein, dipeptidyl peptidase, and AARE might be evolutionally related. The gene was amplified by polymerase chain reaction using two primers. The upper primer (5′-TTTTGAATTCTTACATATGGGCAAGGGGCTTTCA-3′) contained an NdeI I site (underlined), and the lower primer (5′-TTTTGGTACCTTT GGATCCTAAGGGTTTAGCTATCCTTT-3′) contained a BamHI site (underlined). The amplified gene was inserted in pET11a, and BL21(DE3) was transformed by the constructed plasmid. After induction for 4 h at 37 °C, 50 mg of the thermostable 70-kDa protein (as determined by SDS-PAGE) was purified from 2 liters of culture medium. The result of densitometer (data not shown) for SDS-PAGE (Fig. 2) indicated that the purity was about 99%. The purified protein (0.05 mg) in solution was spotted on an Immobilon polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA) and sequenced by a PSQ-1 protein sequencer (Shimazu) at the custom service center of Takara Shuzo. By sequence analysis, the first 20 amino acid residues of the NH2terminus except the NH2-terminal Met were detected. The NH2-terminal sequence was identical to that anticipated from the nucleotide sequence. The extra f-Met residue at the NH2 terminus of the nascent polypeptide, encoded by the initiation codon, was not detected. This shows that the Gly residue neighboring the starting f-Met residue has a small radius of gyration which is essential for the removal of the f-Met residue to yield the mature enzyme (20Levitt M. J. Mol. Biol. 1976; 104: 59-107Crossref PubMed Scopus (907) Google Scholar), and the soluble protein was processed correctly. The high yield of the recombinant protein indicates the very efficient post-translation of the P. horikoshii gene inside E. coli cells, including the removal of the f-Met residue from the nascent polypeptide. It is suggested that the pET system is a good tool for the production of this protein, and the protein has no toxic effect on the growth of E. coli. Unlike other AARE, the protein derived from P. horikoshiineeded a high concentration of NaCl to be dissolved. Therefore, the purified protein solution used for the characterization contained 0.6m NaCl. The molecular mass of the purified protein, as determined by SDS-PAGE (Fig. 2), was consistent with that (72,390 Da) calculated from the amino acid sequences. The molecular mass of the protein determined by HPLC was about 150,000 Da (data not shown). The weighted-average molecular weight measured by light-scattering photometry using the dn/dc value determined for chicken gizzard myosin was about 160,000. Therefore, the protein is likely a dimer structure, instead of the four identical subunits found in mammals (9Mita M. Asada K. Uchimura Y. Kimizuka F. Kato I. Sakiyama F. Tsunasawa S. J. Biochem. ( Tokyo ). 1989; 106: 548-551Crossref PubMed Scopus (46) Google Scholar, 17Kobayashi K. Smith J.A. J. Biol. Chem. 1987; 262: 11435-11445Abstract Full Text PDF PubMed Google Scholar). The absorption coefficient (A280 nm) of the protein at 1% was determined to be 12.0. To examine the activity of this protein, we used Ac-Leu-pNA, Ac-Ala-pNA, Ac-Tyr-pNA, Leu-pNA, and Ala-pNA as substrates. Table I shows the hydrolytic activity (releasing of pNA) of the protein for them. At 85 °C and pH 5.4, the protein exhibited some hydrolytic activity for Ac-Leu-pNA, Ac-Ala-pNA, Ac-Tyr-pNA and no hydrolytic activity for Leu-pNA and Ala-pNA. As shown in Table I, the protein also had hydrolytic activity for acetylpeptides and formylpeptides. Analysis of the products by HPLC revealed that the protein could only release the acylated amino acids from acylpeptides. Therefore, this protein was concluded to be the AARE from the thermophilic archaeon P. horikoshii. The characteristics of this enzyme (hereafter referred to as AAREP) were examined. The optimum pH of AAREP at 85 °C was between pH 4.8 and 5.5 (Fig. 3). The optimum temperature of AAREP at pH 5.4 was about 90 °C (Fig. 4). Its specificity for small substrates was different from those of AAREs in mammals (1Tsunasawa S. Narita K. Ogata K. J. Biochem. ( Tokyo ). 1975; 77: 89-102PubMed Google Scholar, 17Kobayashi K. Smith J.A. J. Biol. Chem. 1987; 262: 11435-11445Abstract Full Text PDF PubMed Google Scholar). Unlike the AARE from rat, AAREP released Ac-Leu better than Ac-Ala from Ac-amino acid-pNA; for most of the substrates used, the specific activity of AAREP was higher than that of AARE from rat (Table I). The activity decreased with increasing the residues of acylpeptides (TableI). Table II shows that AAREP has similar binding affinity for Ac-Leu-pNA, Ac-Ala-pNA, Ac-Ala-Ala, and Ac-Ala-Ala-Ala-Ala. This result is different from that of rat (17Kobayashi K. Smith J.A. J. Biol. Chem. 1987; 262: 11435-11445Abstract Full Text PDF PubMed Google Scholar). The Km value obtained for Ac-Ala-Ala was a little smaller than that for Ac-Ala-Ala-Ala-Ala (Table II). The active site of AAREP seems to be suitable for relatively short acylpeptides. The hydrolytic activity of AAREP toward α-MSH was also examined under the above conditions. The NH2-terminal amino acid sequence of α-MSH was not detected by the protein sequencer after the incubation with AAREP. This result indicates that AAREP cannot release Ac-Ser from α-MSH, unlike the AARE of pig 2K. Ishikawa and S. Tsunasawa, unpublished data. or rat (17Kobayashi K. Smith J.A. J. Biol. Chem. 1987; 262: 11435-11445Abstract Full Text PDF PubMed Google Scholar). It is peculated that AAREP is able to hydrolyze only short acylpeptides.Table ISubstrate specificity of AAREP and HAAREPSubstrate (10 mm)ActivityAAREPHAAREPAARE from Rat (17Kobayashi K. Smith J.A. J. Biol. Chem. 1987; 262: 11435-11445Abstract Full Text PDF PubMed Google Scholar) at 37 °Cμmol/min/mg (enzyme)Ac-Leu-pNA105 ± 11750 ± 8745.3 Leu-pNA<0.01<0.01Ac-Ala-pNA6.9 ± 1.051 ± 0.8457.3 Ala-pNA<0.01<0.01Ac-Tyr-pNA1.9 ± 0.515 ± 1.5Ac-Met<0.01<0.01Ac-Met-Ala344 ± 195,120 ± 8947.1Ac-Ala-Ala478 ± 607,060 ± 507107Ac-Met-Ala-Ala-Ala-Ala-Ala11.7 ± 3.077.0 ± 24Ac-Ala-Ala-Ala-Ala206 ± 461,830 ± 27753Ac-Ala-Ala-Ala-Ala-Ala-Ala13.6 ± 7.8118 ± 20f-Met<0.01<0.01NDf-Met-Ala-Ser1,388 ± 18817,026 ± 295f-Met-Ala97.2 ± 101,513 ± 18242.5f-Met-Leu-Gly694 ± 787,064 ± 550f-Ala-Ala-Ala402 ± 635,363 ± 711f-Met-Ala-Ala-Ala-Ala-Ala9.5 ± 1.666.8 ± 18.7f-Ala-Ala-Ala-Ala-Ala-Ala10.2 ± 2.677.3 ± 14.0The hydrolytic reaction was measured at 85 °C in 50 mmsodium acetate buffer (pH 5.4) containing 0.6 m NaCl and 5% DMF. ND, activity was not detected. Open table in a new tab Figure 4Effect of the temperature on the hydrolytic activity of AAREP (○) and HAAREP (•) on Ac-Leu-pNA. The hydrolytic activity was measured in 50 mm sodium acetate buffer (pH 5.4) containing 0.6m NaCl and 5% DMF. The assay was measured for 10 min.Inset: Arrhenius plot.View Large Image Figure ViewerDownload (PPT)Table IIKinetic parameters of AAREP and HAAREPSubstrateAAREPHAAREPKmkcatKmkcatmms−1mms−1Ac-Leu-pNA11.0 ± 5.5330 ± 3112.9 ± 9.03,200 ± 230Ac-Ala-pNA18.4 ± 4.017.3 ± 0.620.2 ± 5.7144 ± 23Ac-Ala-Ala7.6 ± 0.8790 ± 1558.6 ± 1.09,900 ± 611Ac-Ala-Ala-Ala-Ala13.0 ± 1.5281 ± 4415.2 ± 2.92,090 ± 230The hydrolytic reaction was measured at 85 °C in 50 mmsodium acetate buffer (pH 5.4) containing 0.6 m NaCl and 5% DMF. Open table in a new tab The hydrolytic reaction was measured at 85 °C in 50 mmsodium acetate buffer (pH 5.4) containing 0.6 m NaCl and 5% DMF. ND, activity was not detected. The hydrolytic reaction was measured at 85 °C in 50 mmsodium acetate buffer (pH 5.4) containing 0.6 m NaCl and 5% DMF. Thermostability of the enzyme was examined with CD and DSC. The CD spectrum in the far-UV region of the enzyme was examined at 25 °C and 95 °C. The CD spectrum of the enzyme at 95 °C was a little different from that at 25 °C (Fig. 5). The intensity of the negative ellipticity around 220 nm decreased slightly with increasing temperature. The CD spectrum of AAREP at 95 °C was stable for 24 h. Using DSC from 0 °C to 125 °C, we measured the heat capacity changes of AAREP. We observed two peaks of heat capacity changes of AAREP over 100 °C in the first scan (Fig. 6 A) but no peak in the second scan. Precipitate was observed after the first scan. The temperature of the peaks was independent of the enzyme concentrations examined (0.1–2 mg/ml). This result indicates that the heat inactivation process of the enzyme is irreversible and accompanied by aggregation. The two peaks observed suggest that AAREP consists of two major domains as reported by Miyagi et al. (12Miyagi M. Sakiyama F. Kato I. Tsunasawa S. J. Biochem. ( Tokyo ). 1995; 118: 771-779Crossref PubMed Scopus (20) Google Scholar). These results indicate that incubating AAREP at 95 °C caused its structure to begin unfolding, but the major conformation of the enzyme remained stable from 0 °C to 100 °C. After incubating at 95 °C, we measured the relative activity of AAREP at 85 °C to examine the effect of heating. Incubating at 95 °C appeared to increase the relative activity nearly 7-fold (Fig. 7). The enzyme did not lose its increased activity upon cooling (4–25 °C), suggesting that the activation was irreversible. This heat-activated enzyme (hereafter referred to as HAAREP) was also stable at 95 °C for 24 h. From the light-scattering photometry, the molecular mass of HAAREP was determined to be 260 kDa, and the spatial size of the associated molecule was observed to be expanded in space compared with AAREP; itsz-average radius of gyration (RG) had increased from less than 100 to more than 400. These molecular mass andRG values were virtually constant for nearly 5 days (250–264 kDa and RG = 416–445) at room temperature, without significant decomposition of the molecule or development of aggregation. The molecular mass value indicates that the number of monomers constituting the associated molecule averages a little more than 3 in this condition. However, the significant spacial expansion (RG = 416–445) strongly suggests a conformational change over the whole monomeric structural unit as a result of heat treatment. By heating, the absorbance around 250–280 nm was increased by 10 ± 1.3%, and the intensity of the negative ellipticity of the CD around 220 nm was decreased slightly (Fig. 5). The changes in the absorbance and CD were parallel to the change in the activity of the enzyme. The NH2-terminal sequence of HAAREP remained identical to that of AAREP. The rate of activation by heat treatment was independent of the enzyme concentrations examined (0.04–1.23 mg/ml). Therefore, it is deduced that the conformational change by heat treatment alters the molecular character of monomer and increases the activity. The NH2-terminal section of about 500 residues (12Miyagi M. Sakiyama F. Kato I. Tsunasawa S. J. Biochem. ( Tokyo ). 1995; 118: 771-779Crossref PubMed Scopus (20) Google Scholar) in the enzyme might be related the conformational change by heat treatment. We are continuing to investigate these points. In comparing the characteristics of the two enzymes, we found that HAAREP had a higher optimum pH, above 7.0 (Fig. 3) and a higher optimum temperature, 95 °C (Fig. 4). The relative specificity of HAAREP for substrates was similar to that of AAREP, although HAAREP showed a 7–14-fold increase in specific activity (Table I). The activation parameters of these enzymes were measured from 50–85 °C (TableIII). The temperature dependence on theKm value of Ac-Leu-pNA for AAREP (Km values at 60, 75, 80, 85, and 90 °C were 0.689 ± 0.21, 3.60 ± 0.66, 3.85 ± 0.91, 11.0 ± 6.3, and 18.0 ± 5.6 mm, respectively) was similar to that for HAAREP (Km values at 60, 75, 80, 85, and 90 °C were 0.876 ± 0.24, 4.12 ± 0.51, 3.99 ± 0.66, 12.9 ± 9.0, and 19.7 ± 2.1 mm, respectively) (Table III). From the temperature dependence on the kcat value of Ac-Leu-pNA, the activation energy of HAAREP was found to be greater than that of AAREP (Fig. 4 and Table III). Both ΔS‡ and ΔH‡ values of the activation were increased by heat treatment. The shapes of the DSC curves of HAAREP (Fig. 6 B) and AAREP (Fig. 6 A) were slightly different from each other, but both enzymes seem to be stable below 100 °C. These results suggest that the conformational change in the enzyme by heat treatment has an orienting effect on the catalytic groups of the active site, making the enzyme more active at higher temperatures. It is speculated that HAAREP is a stable intermediate state between the native AAREP state and the heat-inactivated (unfolded) state of the enzyme. Although we have no information about the activity and structure of native AARE in P. horikoshiicells, HAAREP is thought to be the dominant state of the enzyme inP. horikoshii because of the organism's high optimum growth temperature.Table IIIActivation parameters of AAREP and HAAREP for the hydrolysis of Ac-Leu-pNA at 85 °CEnzymeAAREPHAAREPΔG‡ (kcal/mol)16.75 ± 3.315.35 ± 0.26Ea (kcal/mol)15.3 ± 0.825.9 ± 2.9ΔH‡ (kcal/mol)14.6 ± 0.825.2 ± 2.9ΔS‡ (cal/mol/K)−6.0 ± 6.327.5 ± 11ΔG (kcal/mol)−3.21 ± 0.5−3.10 ± 0.8ΔH (kcal/mol)−25.8 ± 2.8−24.6 ± 3.3ΔS (cal/mol/K)−63.1 ± 7.8−60.0 ± 9.3The activation parameters (ΔG‡,Ea, ΔH‡, ΔS‡) and (ΔG, ΔH, ΔS) for Ac-Leu-pNA were calculated using typical Arrhenius plots ofkcat (Fig. 4. inset) and van't Hoff plots of Km, respectively. Open table in a new tab The activation parameters (ΔG‡,Ea, ΔH‡, ΔS‡) and (ΔG, ΔH, ΔS) for Ac-Leu-pNA were calculated using typical Arrhenius plots ofkcat (Fig. 4. inset) and van't Hoff plots of Km, respectively. Until now, AARE has been found only in eukaryotic cells and thought to be related to the initiation of protein synthesis (1Tsunasawa S. Narita K. Ogata K. J. Biochem. ( Tokyo ). 1975; 77: 89-102PubMed Google Scholar, 21Narita K. Tsuchida I. Tsunasawa S. Ogata K. Biochem. Biophys. Res. Commun. 1969; 37: 327-332Crossref PubMed Scopus (25) Google Scholar, 22Liew C.C. Haslett G.W. Allfrey V.G. Nature. 1970; 226: 414-417Crossref PubMed Scopus (71) Google Scholar, 23Yoshida A. Watanabe S. Morris J. Proc. Natl. Acad. Sci. U. S. A. 1970; 67: 1600-1607Crossref PubMed Scopus (36) Google Scholar). The existence of this enzyme in the archaeon P. horikoshiisuggests that the initiation of protein synthesis in archaea is similar to that in eukaryotic cells. From the fact that a number of eukaryotic intracellular proteins are known to beNα-acylated (24Tsunasawa S. Sakiyama F. Methods Enzymol. 1984; 106: 165-170Crossref PubMed Scopus (70) Google Scholar, 25Brown J.L. Roberts W.K. J. Biol. Chem. 1976; 251: 1009-1014Abstract Full Text PDF PubMed Google Scholar), it is speculated that many proteins of archaea are alsoNα-acylated. Furthermore, the existence of proteasomes in P. horikoshii (26Ishikawa K. Ishida H. Koyama Y. Ishimura M. Higuchi K. Kosugi Y. Matsui E. Masuchi Y. Shizuya H. Maekawa A. Matsui I. Nature Biotech. Short Rep. 1997; 8: 63Google Scholar, 27Matsui E. Ishikawa K. Ishida H. Koyama Y. Ishimura M. Higuchi K. Kosugi Y. Masuchi Y. Shizuya H. Maekawa A. Matsui I. Nature Biotech. Short Rep. 1997; 8: 65Google Scholar) suggests that the action of the enzyme AAREP might be related to the ubiquitin/ATP-dependent system of protein degradation (28Hershko A. Heller H. Eytan E. Kaklij G. Rose I.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7021-7025Crossref PubMed Scopus (161) Google Scholar, 29Bachmair A. Finley D. Varshavsky A. Science. 1986; 234: 179-186Crossref PubMed Scopus (1402) Google Scholar, 30Jentsch S. Annu. Rev. Genet. 1992; 26: 179-207Crossref PubMed Scopus (450) Google Scholar, 31Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1210) Google Scholar). Archaea also contains aminoacylase (26Ishikawa K. Ishida H. Koyama Y. Ishimura M. Higuchi K. Kosugi Y. Matsui E. Masuchi Y. Shizuya H. Maekawa A. Matsui I. Nature Biotech. Short Rep. 1997; 8: 63Google Scholar, 32Boyen A. Legrain C. Pierard A. Glansdorff N. Thermophiles 1996. 1996; (conference abstract, 178)Google Scholar), which might play an important role in the recycling of acylamino acids for protein synthesis with the help of AARE. In eukaryotic cells, a strong degree of genetic similarity between AARE and aminoacylase was suggested by Jones et al. (33Jones W.M. Scaloni A. Bossa F. Popowicz A.M. Schneewind O. Manning J.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2194-2198Crossref PubMed Scopus (46) Google Scholar). InP. horikoshii, however, the gene for aminoacylase was found at another locus in the genome (27Matsui E. Ishikawa K. Ishida H. Koyama Y. Ishimura M. Higuchi K. Kosugi Y. Masuchi Y. Shizuya H. Maekawa A. Matsui I. Nature Biotech. Short Rep. 1997; 8: 65Google Scholar), and AAREP did not share homology or activity with aminoacylase. AARE from mammals has been used to removeNα-acylamino acid residues from acylpeptides for protein sequencing at relatively low temperature (37 °C). AAREP may not be used to removeNα-acylamino acid residues of relatively long acylpeptides. However, AAREP is expected to be used for relatively short acylpeptides in sequence analysis at temperatures higher than 90 °C. Studies about the crystal structure, thermostability, and hydrolytic mechanism of AAREP are in progress. We thank T. Hashimoto, M. Jyutori, Dr. Y. Kosugi, Dr. S. Kawasaki, and Dr. S. Tsunasawa for great assistance in the experiments in this study." @default.
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