Gennaro Marino, Gianpaolo Nitti, Maria Immacolata Arnone, Giovanni Sannia, Agata ..... 174,. 38. Zuber, M. (1979) in Biochemistry of Thermophily (Friedman, S.
Vol. 263, No. 25, Issue of September 5, pp.12305-12309,1388 Printed in U.S.A.
CHEMISTRY T H EJOURNAL OF BIOLOGICAL 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterizationof Aspartate Aminotransferase from the Thermoacidophilic ArchaebacteriumSulfolobus solfataricus* (Received for publication, November 2, 1987)
Gennaro Marino, Gianpaolo Nitti, MariaImmacolata Arnone, Giovanni Sannia, Agata GambacortaS, and Mario De Rosa8 From the Dipartimento di ChimicaOrganica e Biologica, Universita’ di Napoli, via Mezzocannone and SIstituto di Biochimica delle Macromolecole, I Facolta’ di Medicina e Chirurgia, via Constantinopoli, 16-80134 Napoli, Italy and Slstituto di Chimica di Molecole di Interesse Bwlogico, Consiglio Nazionale delle Ricerche, via Toiano, 6-80072Arco Felice, Napoli, Italy
Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus, a thermoacidophilic organism isolated from an acidic hot spring (optimal growth conditions: 87 OC,pH 3.5) was purified to homogeneity, The enzyme is a dimer (Mr subunit = 53,000) showing microheterogeneity when submitted to chromatofocusing and/or isoelectric focusing analysis (two main bands having PI = 6.8 and 6.3 were observed). The N-terminalsequence (22 residues) does not show any homology with any stretch of known sequence of aspartate aminotransferases from animal and bacterialsources. The apoenzyme can be reconstituted with pyridoxamine 5”phosphate and/or pyridoxal 5’-phosphate, eachsubunitbinding 1 molof coenzyme. The absorption maxima of the pyridoxamine and pyridoxal form are centered at 325 and 335 nm, respectively; the shape of the pyridoxal form band does not change with pH. The enzyme has an optimum temperature higher than95 “C, and at 100 OC shows a half-inactivation time of 2 h. The above properties seem to be unique even for enzymes from extreme thermophiles (Daniel, R. M. (1986) in Protein Structure, Folding, and Design (Oxender, D. L., ed) pp. 291-296, Alan R. Liss, Inc., New York) and lead to the conclusion that aspartate aminotransferase from S. solfataricus is one of the most thermophilic and thermostable enzymes so far known.
Aspartate aminotransferase (EC 2.6.1.1) is the best known pyridoxal phosphate-dependent enzyme. Two isozymes of aspartate aminotransferase occur in animalcells; one is located in the cytosol, andtheother in the mitochondria. Both isozymes are coded for by nuclear DNA, and only the mitochondrial one, after its synthesis,is exclusively processed and imported into the organelle. Both the amino acid sequence andthe spatialstructure of the twoisozymes have been determined. The above features have been extensively reviewed (1).More recently, aspartate aminotransferase cDNA from several animal sources (e.g. chicken (2), pig (3), rat (4), mouse (5)) has been cloned. Owing to its keyrole in general metabolism, aspartate aminotransferase occurs widely in nature, and it has been assumed that aspartate aminotransferases maybe derived *This work was supported by grants from Minister0Pubblica Istruzione (Rome) (Progettidi Rilevante Interesse Nazionale), Universita di Napoli, and Consiglio Nazionale delleRecerche (Rome) (Progetto Speciale Biotecnologie). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section1734 solely to indicate this fact.
from a common ancestral form. Sequence determination of Escherichia coli aspartate aminotransferase (6) and of the corresponding gene (7) showed nearly 30%similarity between the prokaryotic enzyme and both of the animal isozymes (8). On a more quantitative basis Doonan e t al. (9) observed very similar Kimura constant values (10) (i.e. the average number of amino acid substitutions per site), when comparing the sequence of E. coli aspartate aminotransferase with those of the animal isozymes thus far determined. Hence, at the level of the overall sequence, the cytosolic and mitochondrial proteins areequally related to theprokaryotic enzyme, suggesting that both isozymes originated from a gene duplication in the early eukaryotic cell. Woese and Fox (11)have reclassified all living organisms in three primary kingdoms: eukaryotes, eubacteria, and archaebacteria. The last are prokaryotic organisms and comprise three different phenotypes: methanogens, extreme halophiles, and extreme thermophiles. In order to study further aspartate aminotransferase evolution, the thermoacidophilic archaebacterium Sulfolobus solfutaricus was chosen as a possible source of this enzymatic system. In addition to evolutionary consideration, a study of the aspartate aminotransferase from S. solfuturicus might result in a greater understanding of the almost unknown nitrogen metabolism of these organisms (for a review, see Ref. 12). Further interest stems from potential biotechnological applications and from a more direct approach toward the problem of protein thermostability (13). This paper reports the occurrence of aspartate aminotransferase in S. solfataricus, its purification and its characterization. EXPERIMENTAL PROCEDURES AND RESULTS’ DISCUSSION
Aspartate aminotransferase from S. solfataricus was purified to homogeneity allowing the characterization of the main catalytic and structuralproperties. Portions of this paper (including “ExperimentalProcedures,” “Results,”Figs. 1-3, and Tables I-V) are presented in miniprint at the end of this paper. The abbreviations usedare: AspAT, aspartate aminotransferase; AspATc, cytosolic aspartate aminotransferase; AspATm, mitochondrial aspartate aminotransferase;CSA, cysteine sulfinic acid; DTNB, 5,5’-dithiobis-(2-nitrobenzoic acid); DTT, dithiothreitol;akGlu,2-oxoglutarate; SDS, sodiumdodecyl sulfate; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; SsAspAT, aspartate aminotransferase from s. solfataricus; TEA, triethanolamine; is easily read with TNBS-, 5-thio-2-nitrobenzoate anion. Miniprint the aid of a standard magnifyingglass.Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.
12305
12306
Aspartate Aminotransferase from Sulfolobus solfataricus
The enzyme activity was routinely assayed by using cysteine sulfinic acid as amino acid substrate; however, the results of enzyme specificity studies (Table IV, in the Miniprint), together with otherreports (28) on the identity of cysteine sulfinate with aspartate aminotransferase, allowed us to assign the enzyme activity confidently to L-aspartate aminotransferase. To thebest of our knowledge, this is the firstreport of the existence of this enzymatic activity, so crucial in nitrogen metabolism of eukaryotes and eubacteria (l),in archaebacteria. It is worth noting that theamount of aspartate aminotransferase present in S. solfuturicus, when grown either in minimal or rich media, is comparable with that found in E. coli and yeasts (35), i.e. about 40 pg of enzyme/g of wet cells. The gross molecular properties of aspartate aminotransferase from S. solfutaricus donot differ very much from those of aspartate aminotransferases from animal and eubacterial sources (1). It is a dimer with a subunit molecular weight slightly higher (Mr= 53,000) than that of the other aspartate aminotransferases so far isolated. Furthermore, microheterogeneity can be observed when pure samples are analyzed by chromatofocusing and isoelectric focusing. The observed PI values (6.3, 6.8) are close to those of cytosolic isozymes from animal sources (8). The amino acid composition, when compared with that of the enzyme from mammalian, avian, and eubacterial sources (Table 111, in the Miniprint), shows, notably, that aspartate aminotransferase from S. solfutaricus has an unusually high lysine content (43 mol/mol subunit) and a low content of histidine residues (2 mol/mol subunit). It is worth noting that 3 histidine residues appear to be invariant in aspartate aminotransferases from mammalian, avian, and eubacterial sources so far sequenced and are located in the network of the active site (36, 37). It will be particularly interesting to investigate the location of the 2 single histidine residues in aspartate aminotransferase from S. solfataricus. As for the high lysine content, it should be noted that it is balanced by a lower arginine content. Therefore, the suggestion made by Zuber (38) that thermophily is related to the replacement of lysine with arginine residues does not seem applicable to 5'. solfataricus aspartate aminotransferase.Comparison of amino acid composition of aspartateaminotransferases does not agree also with the data reported by Singleton et ul. (39) who found that Asx and Ser are decreased in thermophilic proteins, whereas Arg is increased. We found that only the average hydrophobicity (H@),one of the macroscopic parameters derived from the amino acid composition indicative of thermophilic proteins (40), couldbe correlated with the thermal stability of aspartate aminotransferase from S. solfaturicus (Table 111, in the Miniprint). However, we believe that not even this parameter can adequately account for the observed high thermostability shown by aspartate aminotransferase from S. solfutaricus (see below). The N-terminal sequence (22 residues) does not show any significant homology with any sequence length, or even with the presequence, of aspartate aminotransferases so far determined (2, 3, 6). The sequence data allowed us to construct oligonucleotide probes to screen a S. solfaturicus genomic library (41) with the aim of determining the entire primary structure of aspartate aminotransferase from S. solfuturicus from the gene sequence. Addition of pyridoxamine phosphate and 2-oxoglutarate after each purification step was necessary to recover the enzyme activity fully. The holoenzyme can be resolved at slightly acidic pH in the presence of sulfate ions, and a fully active enzyme is obtainedafter addition of pyridoxamine
phosphate or pyridoxal phosphate. A Scatchard plot of titration data shows that 1 mol of aspartate aminotransferase from solfuturicus binds 2.3 mol of pyridoxal phosphate, i.e. 1:1 coenzyme/subunit stoichiometry, with a K d = 5 x M. The absorption bands of the coenzyme in the pyridoxal and pyridoxamine forms are ipsochromically shifted to 335 and 325 nm, respectively, when compared with the absorption maxima exhibited by aspartate aminotransferases from other sources (1). More precisely, the pyridoxal form, obtained either by incubation of the enzyme with pyridoxamine phosphate and 2-oxoglutarate or by addition to the apoenzyme of pyridoxal phosphate, shows an asymmetrical band with a maximum centered at 335 nm and a shoulder at about 360 nm (Fig. 2, in the Miniprint). The pyridoxamine form, obtained by the addition of an excess of cysteine sulfinic acid, shows a symmetrical band centered at 325 nm. Furthermore, most surprisingly, the band of the pyridoxal form does not change with pH. As the enzyme is inactivated hy sodium borohydride it can be assumed that the usual internal aldimine is formed from the carbonyl group of the coenzyme with an amino group of the apoprotein. It is interesting to note that aspartate aminotransferase from S. solfataricus shows a broad range of optimum pH, and itis fully active also at pH 5.8. At this pH no absorption at 430 nm, due to theprotonation of the aldimine bond, is observed. A broad range of optimum pH has also been reported formitochondrial aspartate aminotransferase in contrast with the narrower one observed for cytosolic aspartate aminotransferase(42). Michuda and Martinez-Carrion (42) demonstrated that different pH activity profile was due to different binding of ketosubstrates. However the pK, of the active site chromophore of cytosolic aspartate aminotransferase and of mitochondrial aspartate aminotransferase is very similar, i.e. pKo = 6.3. To explain both spectral properties and activity pH dependence, it could be assumed that the active site chromophore of aspartate aminotransferase from S. solfutaricushas ananomalously low apparent pK.. Although further structural investigation is needed to clarify this point, the present results suggest that the environment of the coenzyme binding site might be significantly different from that of the other aspartate aminotransferases. Aspartate aminotransferase from S. solfataricus, although possessing the classical ping-pong mechanism of transamination (34), displays kinetic properties and substratespecificity (Table IV, in theMiniprint) that appear unique. Activity of aspartate aminotransferase from S. solfataricus increases steadily in the range of temperature we were able to investigate, i.e. from 25 to 95 "C. The aspartate aminotransferase from S. solfutaricusoptimum temperature is thus higher than 95 "C, demonstrating the extreme thermophilic character of this enzyme. Like several other thermophilic enzymes (43-45), aspartateaminotransferase from S. solfaturicus shows a thermal transition in conformation as indicated by the break in anArrhenius plot around58 "C (insert to Fig. 3, in the Miniprint). Furthermore the enzyme is fully stable at 90 "C for at least 6 h, and shows a half-inactivation time of about 2 h in sealed vials at 100 "C. The above properties are quite unusual even for enzymes from extreme thermophiles (46). Therefore aspartate aminotransferase from S. solfaturicus can be considered a very interesting model for the study of structural requirements necessary to achieve high thermophily and thermostability inenzymatic proteins.
s.
Acknowledgments-We acknowledge Lucia Albano and Giuseppina Travaglione for assistance in sequence and amino acid analyses. We also acknowledge Enrico Eaposito and Valeria Calandrelli for their technical assistance.
Aspartate Aminotransferasefrom Sulfolobus solfataricus REFERENCES Christen,P., and Metzler, D.E. (1984) Transaminases, John Wiley & Sons, New York Jaussi, R., Cotton, B., Juretii, N., Christen, P., and Schumperli, D. (1986) J. Biol. Chem. 2 6 0 , 16060-16063 Joh, T., Nomiyama, M., Maeda, S., Shimada, K., and Morino, Y. (1985) Proc. Natl. Acad. Sci. U. S. A. 8 2 , 6065-6069 Horio, Y., Sakakibara, R., Tanaka, T., Taketoshi, M., Obam, K., Shimada, K., Morino, Y., and Wada, H. (1986) Biochem. Biophys. Res. Commun. 134,803-811 5. Obam, K., Nomiyama, M., Shimada, K., Nagashima, F., and Morino, Y. (1986) J. Biol. Chem. 261,16976-16983 6. Kondo, K., Wakabayashi, S., Yagi, T., and Kagamiyama, M. (1984) Biochem. Biophys. Res. Commun. 122, 62-67 7. Kuramitsu, S., Okuno, S., Ogawa, T., Ogawa, H., and Kagamiyama, H. (1985) J. Biochem. (Tokyo) 97,1259-1262 8. Kagamiyama, H., Kondo, K., and Yagi, T. (1984) in Chemical and Biological Aspects of Vitamin Be Catalysis (Evangelopulos, A. E.,ed) PartB, pp. 293-302, Alan R. Liss, Inc., New York 9. Doonan, S., Martini, F., Angelaccio, S., Pascarella, S., Barra, D., and Bossa, F. (1986) J. Mol. Euol. 2 3 , 328-335 10. Kimura, M. (1983) The Neutral Theory of Molecular Evolution, Cambridge University Press, Cambridge, United Kingdom 11. Woese, C. R.,and Fox, G. E. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,5088-5090 12. Jones, W. J., Nagle, D. P., and Whitman, W. B. (1987) Microbiol. Rev. 61,135-177 13. Imanaka, T., Shibazaki, M., and Tavagi, M. (1986) Nature 3 2 4 , 695-697 14. De Rosa, M., Gambacorta, A,, and Bulock, J. D. (1975) J. Gen. Microbiol. 8 6 , 154-164 15. De Rosa, M., Gambacorta, A., Nicolaus, B., Giardina, P., Poerio, E., and Buonocore, V. (1984) Biochem. J , 224,407-414 16. Tsang, V. C. W., Peralta, J. M., and Simons, A. R. (1983) Methods Enzymol. 92,377-391 17. Ricci, G., Dupre', S., Lo Bello, M., Achilli, M., and Federici, G . (1982) ZRCS Med. Sci. 10,672-673 18. Moore, N. S., and Stein, W. H. (1963) Methods Enzymol. 6,819831 19. Him, C. H. W. (1967) Methods Enzymol. 11, 197-199 20. Penke, B., Ferenczi, R., and Kovacs, K. (1974) Anal. Biochem. 60,45-50 21. Pucci, P., Sannia, G., and Marino, G. (1983) J. Chromatogr. 2 7 0 , 371-377 22. Reisfeld, R. A., Lewis, U. J., and Williams, D. E. (1962) Nature 196,281-283
SUPPLEMENTAL MATERIAL TO
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23. Davis, B. J. (1964) Ann. N . Y. Acad. Sci. 121,404-427 24. Weber, K., Pringle, J. R., and Osborne, M. (1972) Methods Enzymol. 26,3-27 25. Di Donato, A., Fiore, R., Garzillo, A. M., and Marino, G. (1983) FEBS Lett. 163.93-102 26. Bradford, M. M. (1976) Anal. Biochem. 7 2 , 248-254 27. Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275 28. Recasens, M., Benezra, R., Basset, P., and Mandel, P. (1980) Biochemistry 19,4583-4589 29. Lowe, P. N., and Rowe, A. F. (1985) Biochem. J. 232,689-695 30. McEvily, A. J., Flint, A. J., and Morrison, J. M. (1985) Anal. Biochem. 1 4 4 , 159-164 31. Bigelow, C. C. (1967) J. Theor. Biol. 1 6 , 187-251 32. Gianfreda, L., Marino, G., Palescandolo, R., and Scardi, V. (1974) Biochem. J. 137,199-203 33. Mavrides, C., and Orr, W. (1975) J. Biol. Chem. 250,4128-4133 34. Velick, S. F., and Vavra, J. (1962) J. Biol. Chem. 237,2109-2122 35. Porter, P. B., Barra, D., Bossa, F., Cantalupo, G., Doonan, S., Martini, F., Sheehan, D., andSusan, M.W. (1981) Comp. Biochem. Physiol. 6 9 B , 737-746 36. Hayashi, H., Horio, Y., Tanaka, T., Taketoshi, M., and Wada, H. (1987) in Biochemistry of Vitamin B6 (Korpela, T., and Christen, P., eds) pp. 39-42, Birkhuser Verlag, Basel 37. Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, M., and Christen, P. (1984) J. Mol. Biol. 1 7 4 , 497-525 38. Zuber, M. (1979) in Biochemistry of Thermophily (Friedman, S. M., ed) pp. 267-285, Academic Press, New York 39. Singleton, R., Jr., Middaugh, C. R., and MacElroy, R. D. (1977) Znt. J. Peptide Protein Res. 10, 39-50 40. Merkler, D. J., Farrington, G. K., and Wedler, F. C. (1981) Znt. J. Peptide Protein Res. 1 8 , 430-442 41. Cubellis, M. V., Rozzo, C., Sannia, G., Arnone, M. I., and Marino, G. (1987) in ChemicalandBiological Aspect of Vitamin Catalysis (Korpela, T., and Christen, P., eds) pp. 125-128, Birkuser Verlag, Basel 42. Michuda, C. M., and Martinez-Carrion, M. (1970) J . Biol. Chem. 246,262-269 43. Zuber, M. (1981) in Structural and Functional Aspects of Enzyme Catalysis (Eggerer, M., and Huber, R., eds) pp. 114-127, Springer Verlag, Berlin, Heidelberg, New York 44. Fabry, S., and Hensel, R. (1987) Eur. J. Biochem. 166,147-155 45. Sugimoto, S.,and Nosoh, Y. (1971) Biochim. Biophys. Acta 236, 210-221 46. Daniel, R.M. (1986) in Protein Structure, Folding, and Design (Oxender, D. L., ed) pp. 291-296, Alan R.Liss, Inc. New York
Aspartate Aminotransferase from Sulfolobus solfataricus
12308
-
Antibodies and Imunoadsorbent PreDaration Polyclonal anti-AbpAT a n t i b o d i e s v e r e =pared by Immunlzatlon of New Zealand white rabbrtr, twice wlth 0.25 mg Of Durified aspartate aminotransferase and then three times With d smaller amount of antigen 1 5 0 ugl, at intervals of 15-20 days. Purified lmmunogiobulins were prepared from serum after fractionation i n 45% ammonium sulphate and on Protem A - SeDharose. SDeciflatv and purificatmn
ImunodfflnltY chramatoaraphy - Active fractions eluted from the QSepharose Fast Flow column were pooled, dialyzed against 100 mM potassium phosphate IpH 6.91and applied to theimmmlOad60Tbent-sephlrose column 10.7~4 mi equllibrated ~n the same buffer. The column was washed with 20 ml of same buffer containing 2 M KC1. Enzyme was Starting buffer and then with the ?l,ltedwitn 50 m succinate buffer (pH 3 . 0 ) containing 0.1 mM pyridoxamine 5 -P. 5 mM akG1u. Active fractmns were pooled and dlalyzed against 5 0 mM TRIS-HC1 (pH 7.51.
m
-
d m The enzymatic activity was rourlnely assayed a t 60 O C . The rate Of increase in absorbance at 412 m due to the addition Of the enzyme to a reaction mixture 12mll containing 2 mM 2-oxoglufarateand 13 m L-cysteine sulfinate, in 50 mM TRIS-HC1 [pH 8.51. 0.1 mM EDTA, and 0.15 mM DTNB was monitored. one unit Of enzyme activity 15 defined as the amount Of enzyme that catalyzes the transamination of 1.0 mol Of rubsrratelmin, under were calculated from the molar extinction the condition speclfied. units Coefficient of 5-thio-2-nltrobeneoate formed during the reaction (171. The effects Of pH and temperature on the aCtlvLty were studied by pzemcubating the purifred enzyme under the reported Conditmns and then assaying the residual activity at TO determine kineticparametersthe 60 'c. concentrations of akGlu and CSAwere varied ~n the assay.
In order c o evdluate substrace specificity, the purifiedenzyme 132 m ) was incubated for various time periods 110, 30 and 60 mint a t 60 -c ln 100 m~ TEA-HC1 (pH 8.01 In the presence of 5 mM amino acid and 5 mM akG111. In the case of glutamate, oxaloacetate (20 mMI as amino acceptor and Shorter time periods 1 2 , 5 and 10 minl were used. The reaction was quenched by addition of 20% trIChloZOaCetlC acid at 0 'C for 10 m l n , neutralized wlth 6 N KOH and centrifuged at 10000 g for 10 min. The supernatant was recovered and amino acid Concentration was determined by amino acid analysis. Activities toward CSA were Cake" as 100%. Rmino acid anaiVris- Mlno acid analyses were carried D U ~on a LKB aminoacid analyzer. Protein samples (25-50 iigl were hydrolysed & 110 'C for 24. 48 and 72 hrs, in 6 M HC1 1181. Cysteine residues determined after performic acid Oxidation (191. Tryptophan residues determined after hydrolysis in vacuo at 110 DC for 24 hrs in 3 mercaproethlnsulfoniC acid 1201.
FIG. I , ElutionprofileofQ-SepharoaeChrOmatogrnphiCstep zn the purification procedure Of SsAspAT. 2.5~30cm column equilibraced with 20mM Gly-NaOH (pH 9.01 and eluted wLth 1 1 0-0.5 M NaCl linear sradient in the same buffer. Flow rate was 60 mllh.
the present conditions, recoveries obtained from Affi-Gel Blue Chromatography very poor; therefore when p~lyclonalantibodies were available (see below] a singleimmunoaffinitystep was perfomed afterQ-sepnarose separation (Table 111.
were
4400 at were were N-O-
~Titratlo" Of cysteine resldues - Enzyme samples ml, 10.25 0.1 mg/mll were treated with 0.75 ml Of 1 mM DTNB ~n 0.1 M sodium phosphate(pH 7.61 in the presence or absence Of SDS. The reactionwas monitored SpeCt.OphotOmet~~CLIly by measuring the~ n c r e a s ein absorbance Dit 412 m. NH,-temlnal analysis - NH2-terminal sequence analysis was performed on a Beckman model 890 C sequencer, rnodlfled wlth a cold trap. urlng a 0.2 M as carrler. Phenylthiohydanfoin derivatives Quadrol program and polybrene were xdencified byreverse phase high performance llquid chromatography Using d Beckman liquid chromatograph,mcdel 332, as described 121).
-
Other methods Disc-gel electrophoresis was perfmrned at 7.5% or 10% acrylamide, both at acid 1221 and alkaline 1231 pHs. SDS-PAGE was performed as described by Weber et a l . I241 on 7.5-15% acrylamide gels. Analytical isoelectric focusing, ln the pH range 5.0-7.5, Was performed on a Multiphor apparatus fromLKB Istackholm. Sweden1 polyacrylamide-gel slab in fallowing the manufacturer's instructions. Relatlve molecular weightofnative e n l m e was determined by gel filrratlon on a 2orbe.X GF-250 1 0 . 9 4 ~ 2 5 cml C o l m (eluted with 0.2 PI oocassiumphosphate, PB 7.0 at a flowrateof 1 mllminl. M, under dissociatini coiditions~wasdetermined by SDS-PAGE. 11x18 Cm) Of PolYbuffer ChrOmatofocuSing was performed on a COlexchanger equilibiared in 25 mM imidazole-HC1 buffer(pH 7.4) at 4 'C. The column was washed with 4 0 ml of Starting buffer and then eluted with 160 ml Of a ~olutionII;8I of Polybuffer 74 [pH 5.01, at a flow rate of 24 mllh. Enzymatic activity andpH were determined an collected fractions.
The holoenzymewas resolved essentiallyaE already reported 1251. was determined by the Bio-Rad Protein Assaysyscem Protein concentration 1261, or by the Lowry method 1271. uslng bovine serum albumin as a standard.
RESULTS
-
Enzymatic accivity was routinely determined a t 60 *c by using cysteine Sulfinateab c4 substrate in the presence of DTNB and by following DTNB by sulphite 1281. the formation of TNBS-. due to the reduction of
ASSay
assay of aspartate with CSA allows a Very sensitive Substitution in the direct detection of enzymatic activity and o v e ~ c o m sthe problem of thermal assay conditions used,1i.e. PH 8 . 5 degradation of oxaloacetate Which, in the at 60 OC1. showed a I4 min half-life (data not shovnl. TABLE I PUrifiCatlOn
Of
AspAT
from
200 g Of wet cells Of Sulfolobus
SPECIFIC TOTAL TOTAL STEP PURIFICATION YIELD PROTEIN ACTIVITY ACTIVITY lmgl IUI 566
TABLE I l l
mlno acld composition and average hydrophobicity IHmI Of SSADPAT compared With those of the enzymefrom pig hearc, chicken heartand E- co11
5240 extract Crude Heatmg and INHllZS04 frac.
1%) 100
86
490
1
2
Q-Sepharore
519
415
0.8
73
8
5-Sephawse
133
358
2.7
63
27
45
380
41
490
15
840
Affi-Gel Blue I
6.7
255
Ultroqel
4.749
230
Affi-Gel Blue I 1
1.0
84
38
84
PUriflCatlOn - The purlfication procedure of Aspartate aminotransferase I. fromBolfataricus grown in minimal medlum is reparted in Table The yieldt-, with a 800 fold purification. The homogeneous enzyme shows a speclfic activityof 8 6 - 1 0 0 ulmg. Anion exchange Chromdtogrdphy using Q-Sepharose Splits the enzymatic (AT-I and AT-11, In Fig. 1). Further distinctpeaks dctivity intotwo purification was carried out only on the more abundant active fractLon. TO homogeneous protein it was essential to perform Affl-Gel Blue obtain a CnrOmbtOgr&phy. Such a step has also been used in recent pUrlf1CLtlOn manmallan sources 129, 30). However, under protocolsforAspATfrom
S.solfa-
Chicken" piga
taricus cit
m ~ t cit
mi=
E.coli'
mollmol Subunit
ma11100 mol
3.60 0.45 9.68 6.08 3.60 7.88 7.43 1.80
I FOLDI
(U/mgl
0.1 2990 0.2
SOlfatariCuI
s.solfa-
taricuS aminoacid
24 16 25 20 26 2 43 27 16 35 33 8
1.35 7.66 5.63 6.76 14.07
6 34 25
10.58
47 28 26
6.31 15.86 11.26 0.90
30 18
50
4
22 6
Aspartate Aminotransferase from Sulfolobus solfataricus
12309
Edman degrldation was performed on two dlfferent proteinsamples using a h w i d phase sequencer. The resulting HH2-tetminal sequence in the followina: 5 10 HH2-V=1-Ser-Le~-Le~-A~P-Ph=-A=~-Gly-A~"-M=t15 20 SeT-Gln-V~1-ThT-Gly-Glu-Thr-Thr-Leu-Leu-
Tyr-LyP
-....
-
Coenznne bindin The enzyme sh-8. at pH 5.0 and pH 8 . 0 , in the absence or w e ence of 2-Zxog1ute.r te ab80 bance maxim centered at 280 nm (EM = 1 . 2 ~ 1 0 ~ 1 and , at 3 3 5 nm (E8 ='l.OxlOi). Addition of excess glutamate shifts the latter peak to 325 nm (Fig. 2). After reduction by addition ofa 200-fold molar excess Of NaBH the absO1ption peak due to the active site-bound coenzyme shifts to 315
plots Show that SsASPAT catalyzes Other aminotransferases 1 3 4 1 .
a pini-pow riaction
as demonstrated for
%.
TABLE IV
I5 ml by Relatlve activlty of SsAspAT towards dlfferent minoacid Substrates using 2-onoglutarare ( 5 mMt as amino acceptor. Activity was assayed et 60 OC In 100 mM TEA-HC1 (pH 8 . 0 1 by measurinq the amountOf the reeldual amino acid substrate and that of glutamate f a m e d by amino acid analysis. 1n the case of glutamate a higher m l n O acceptor concentration ( i e DXalOacetate 20 M I 1 and shorter incubation times (2.5 and 10 minl were used. Activities toward CSA were taken an 100%. For comparison the relative activity Of AnpAT from eubacterial and mamnalian 90urceSr as reported in 181. are Shown.
CSA
100 74 185
100
100
(a
27 77
25 1.1
"
0.9 0.9
"
4.8
0.4
1.2
0.007 0.005
8.5
0.05
. .
0.0
"
loa 29 87 ~~
0.11 0.04
0.17 "
TABLE V determined for ShAspAT. Activity has been Apparent KM for CSA and &Gl; determined as descrlbed under experimental" at 60 *C in the presence of different concentration Of CSA o r & G l U . For comparison the apparent KM Of ABpAT from eubacterill and mamnalian soUI.cel are reported.
K ~ x l 0M ~ S.aolfataricus FIG. 2. Absorption Spectra of SsASpAT 12.1 m g l m l ) in 5 0 W4 TEA-HC1 buffer pH 8.0 (curve 1 1 ; plus 1.0 mM (curve 21; sodium is glutaMte borohydrlde treated enzyme in 200 mM 6 . 5 (curve 3 ) . sodium phosphate pH The amplified visible-region Of the spectra is reported in the insert.
FIG. 3. Temperature dependence Of aCtivltY O f SSASPAT. Airrhenius Plot oftheresults in the range 25-95 'c Shorn the insert.
The enzyme is fully stable when incubated at 90 *C. even after 6 hours in the Presence of 0.1 W4 PyIidoXmlne 5"P. 2 mM &Glu and 1 mglml ESA in 5 0 mM TRIS-HC1 buffer IpH 7.51. Under the above conditions. at 100 .c, in sealed V 1 a 1 6 , SEABPAT Shows a half-inactivation tlme of about 2 hours.
CSA
1.8
akGlu
0.3
cit pig"
0 .24
mit pig"
22.6
3.4
1.0
1.1
