Purificatioii and Some Properties of Threonine Dehydratase from Yeast

Transcription

1 Eur. J. Biochem. 24 (1971) Purificatioii and Some Properties of Threonine Dehydratase from Yeast Tsunehiko KATSUNUMA, Sigrid ELSASSER, and Helmut HOLZER Biochemisches Institut der Universitat, Freiburg im Breisgau and Forschungsgruppe fur Biochemie der Gesellschaft fur Strahlen- und Umweltforschung (Received August 23/September 29, 1971) Biosynthetic L-threonine dehydratase was purified from bakers yeast and crystallized. The enzyme was homogeneous as judged by ultracentrifugation and electrophoresis. SZO, was 9.2 S, and the molecular weight was estimated to be approximately The enzyme exhibits a slight yellow color. An absorption maximum in the visible region was at 410 nm. Apparent Michaelis constants for L-threonine and L-serine are reported. Threonine dehydratase catalyzes the first reaction of the biosynthetic sequence leading from threonine to isoleucine. Inhibition of the enzyme from Esherichia coli by isoleucine provided Umbarger [ 1,2] a prototype for the concept of end-product inhibition as a general regulatory mechanism. Threonine dehydratase from yeast, like that from E. coli, is inhibited by isoleucine and activated by valine [3]. Furthermore, enzymatic activity in crude extracts is stabilized by pyridoxal phosphate, protected by threonine, valine and isoleucine against inactivation upon dilution, and enhanced by ammonium ions [3-61. Prerequisite to the further investigation of the regulatory properties of this enzyme is its availability in pure form. This paper reports a procedure for 1300-fold enrichment and crystallization of the enzyme from bakers yeast. The crystallized enzyme behaves homogeneously upon sedimentation and electrophoresis. Some physical and kinetic properties of the enzyme are described. A preliminary communication of these results has appeared [7]. MATERIAL AND METHODS Materials Bakers yeast (Pleser Hefe, Darmstadt-Eberstadt) was obtained from BAKO (Backer-Einkauf E.G.M.B.H. Freiburg i. Br.). Pyridoxal 5-phosphate, L-threonine, L-serine, L-homoserine, L-isoleucine, L-valine, and bovine serum albumin were obtained from Sigma Chem. Corp. (St. Louis, U.S.A.), NADH, and lactate dehydrogenase from Boehringer Mannheim GmbH (Mannheim Germany), protamine sulfate and glass beads (d = mm) from Carl Roth OHG (Karlsruhe), and Sephadex G-200 from Pharmacia (Uppsala, Sweden). Enzyme. Threonine dehydratase or L-threonine hydrolyase (deaminating)(ec ). Nethods Threonine dehydratase activity was determined using a coupled optical test with NADH, and lactate dehydrogenase described by Holzer et al. [3,5]. The standard buffer used in the purification procedure contained 0.05 M potassium phosphate ph 7.6, 1 mm pyridoxal 5-phosphate, 0.1 mm EDTA, 1 mm mercaptoethanol, and 50 pm L-valine. Protein concentration was determined according to Lowry et al. [S] using bovine serum albumin as reference. One unit of enzymic activity is the amount producing 1 pmol 2-oxobutyrate per minute under the test conditions of Holzer et al. [3,5]. Specific activity is expressed as units per mg protein. Disc electrophoreses was carried out in the Buchler Polyanalyst apparatus (Buchler Instruments,Chicago, U.S.A.). The anionic gel-system of the Buchler Instruction manual was used. Sedimentation studies were made in a Spinco model E ultracentrifuge at 4.6 C and rev./min, using a 12 mm aluminium singlesector centerpiece and a phase-plate angle of 70 C. Determination of sedimentation coefficient and its correction to standard conditions followed established procedure [9]. Sedimentation equilibria were measured according to Yphantis [lo] using ultraviolet optics. Molecular weights were calculated from the equation 2 RT d In A280 M, = w2(1 - G@) d r2 were d In A,,,/d r2 is the slope of a plot of In AZBO with respect t)o r2, R is the gas constant, T is the absolute temperature, e is the density of the solvent, (1) is the angular velocity of the rotor, and 6 is the partial specific volume of the protein, assumed to be ml/g as calculated for threonine dehydratase from E. coli [ill and Bacillus subtilis [la]. 6

2 84 Threonine Dehydratase from Yeast Eur. J. Biochem. Absorption spectra were taken with a Cary-15 spectrophotometer. An apoenzyme was prepared by dialyzing the purified enzyme against 500-fold volume of 0.1 M phosphate buffer ph 7.0 containing 0.1 mm NH,OH for 1.5 h, followed by addition of an equal volume of saturated ammonium-sulfate. The precipitate was collected by 10-min centrifugation at x g, washed with half-saturated ammonium sulfate, again centrifuged, and finally dissolved in 0.1 M phosphate buffer ph 7.0. Thus prepared, the apoenzyme showed about 300/, of the activity obtained upon saturation with pyridoxal 5-phosphate ; the apparent Michaelis constant for pyridoxal5-phosphate was 3 pm. RESULTS Purification of Threonine Dehydratase The procedure for purification of the enzyme is summarized in Table 1. All operations were performed at 0-5 "C. Step 1. Extraction from Cells, 2 kg of bakers' yeast were suspended in 6 1 of standard buffer, and 100-ml portions vibrated together with I00 g of glass beads for 5 min (Vibro Mischer E 1, Chemap AG MannedorfIZH, Switzerland). The supernatant obtained by centrifugation at xg for 30 rnin comprised the crude extract. Step 2. Protamine Sulfate Treatment. To 6 1 of crude extract, 200ml of freshly prepared 3.0 /, protamine-sulfate solution was added with stirring. After 10 min the mixture was centrifuged for 30 rnin at x g and the precipitate was discarded. Step 3. First Ammonium-Sulfate Fractionation. To the supernatant solution from step 2, 390g of solid ammonium-sulfate were added per liter with stirring. After 20 min the mixture was centrifuged at x g for 10 min, and the precipitate was washed with 500 ml 1.1 M K,HPO, and recentrifuged. The precipitate was dissolved in 300ml of standard buffer. Step 4. First Heat Treatment. The above solution was made 10 mm in L-isoleucine, divided into thirds, and swirled in a 70 "C bath until its temperature reached 60 "C. It was then immediately chilled in an ice-water bath and centrifuged at x g for 10 min. Step 5. Second Ammonium-Xulfate Fractionation. 313 g of solid ammonium-sulfate were added per liter of supernatant from step 4 ; after 20 min the suspension was centrifuged at x g for 10 rnin and the precipitate washed with 50 ml 1.1 M K,HPO,. The precipitate was dissolved in standard buffer and freed from ammonium sulfate by gel-filtration on Sephadex G-75. The turbid eluate was centrifuged for 60 min at x g. Step 6. Third Ammonium-Sulfate Fractionation. To each liter of supernatant was added 158.4g of solid ammonium-sulfate and after 20 min the precipitate was removed by centrifugation at x g for 10 min and discarded. Additional solid ammonium-sulfate (53 g per liter) was added to the supernatant and the precipitate was collected by centrifugation at x g for 10 min. The precipitate was dissolved in 15 ml standard buffer containing 10 mm L-isoleucine. Step 7. Second Heat Treatment. Treatment in step 4 was repeated. Subsequently the enzyme was precipitated with ammonium sulfate (196 g/l), centrifuged at xg for 10 min and redissolved in 5 ml standard buffer. Step 8. Exclusion Chromatography on Sephadex G-200. The solution of step 7 was applied to a column (2.5 x 100 cm) of Sephadex G-200 equilibrated with standard buffer. The flow rate was 20 ml/h. Threoninedehydratase activity emerged in the second of two protein peaks. Fractions containing enzymatic activity were combined and dialyzed against 100-fold volume of standard buffer saturated with ammonium sulfate for 60 min. The precipitated protein was spun down at x g for 10 min and redissolved in 3 ml of standard buffer containing 10 mm L-isoleucine. Step 9. Crystallization. The solution from step 8 was made up to an ammonium-sulfate concentration of 140g per liter and centrifuged for 10 min Table 1. Purification of yeast threonine dehydratase Step Volume Protein Speciflc activity Purification Crude extract Protamine-sulfate supernatant First ammonium-sulfate precipitate First 60 "C supernatant Second ammonium-sulfate precipitate Third ammonium-sulfate precipitate Second 60 "C supernatant Gel filtration on Sephadex G-200 First crystallization Second crystallization ml unitslmg fold Recovery "a

3 Vol.21, No.1, 1971 T. KATSUNUMA, S. ELSASSER, and H. HOLZER 85 Fig. 1. Photomicrograph of crystalline threonine dehydratase ( x 2000) at x 9. The precipitate was discarded and the supernatant kept at room temperature. After about 10 min, crystals appeared. Step 10. Recrystallization. The first crystals were gathered by centrifugation at 3020 xg for 10 min and redissolved in 1 ml of standard buffer containing 10 mm L-isoleucine. Finely powdered ammoniumsulfate was added gradually until a sight turbidity was evident. The solution was placed in ice-bath and allowed to come slowly (about 12 h) to room temperature. After standing an additional 4 h at room temperature crystals appeared. The suspension was centrifuged at 3020 x g for 10 min and the precipitate was washed with ammonium-sulfate solution (100 g/l) at room temperature. The crystals shown in Fig. 1 were obtained by centrifugation at 3020 x g for I0 min. Specific activity of the first crystalline preparation was approximately 108 units per mg protein. There was no increase in the specific activity after recrystallization. In the absence of pyridoxal 5-phosphate and L-valine or L-isoleucine, the purified enzyme was very unstable as described earlier for partially purified enzyme preparations [3,4,13]. Full activity was retained in standard buffer for two weeks at 5 "C. Homogeneity of Purified Enzyme The crystallized enzyme, dissolved in standard buffer, sedimented homogeneously in the analytical centrifuge (Fig. 2). Disc electrophoresis in polyacrylamide gel disclosed a single protein band (Fig.3). The homogeneity of the enzyme was also examined in cellulose-acetatemembrane electrophoresis. At ph 7.0 and 8.0 only a single band was observed. Molecular Weight All sedimentation experiments were performed in 0.05 M potassium-phosphate buffer ph 7.6 in the presence of 0.5 mm L-valine to ensure enzyme stability. The sedimentation constant s,20 was found to be 9.2 S. The molecular weight of the enzyme was calculated from high-speed sedimentation-equilibrium measurements by the method of Yphantis [lo]. The molecular weight extrapolated to zero protein concentration was found to be (S.D. = 9500) (Fig. 4). Absorption Xpectra A concentrated solution of the enzyme looks yellow, corresponding to an absorption maximum in the visible region at 410 nm (Fig. 5) which is typical of enzymes containing pyridoxal 5-phosphate as a hydrogen-bonded Schiff base. In the ultraviolet region, an absorption maximum was observed at 277 nm. The absorbance ratio at 280:260 nm was 1.6 and at 280:410 nm was about 6. ph Optimum of the Reaction Rate The ph optimum for the deamination of L-threonine was determined by assaying enzyme activity in

4 86 Threonine Dehydratase from Yeast Eur. J. Biochenr. Fig. 2. Ultracentrifugal sedimentation patterns of threonine dehydratase. The protein concentration was 5 mg/ml in standard buffer. The photographs were taken at intervals of 16 min after reaching rev./min, at a phase plate angle:of 70, rotor temperature was 4.6 "C. Direction of sedimentation is from left to right A B I' Origin Fig. 3. Polyacrylamide-gel disc-electrophoresis of threonine dehydratase. The protein was applied to 3.0 /, polyacrylamide gel equilibrated with 375 mm Tris-HCI ph 9.3 and subjected to 2.5 ma per column (75 x 5 mm) for 100 min at room temperature. Acrylamide was photopolymerized in the presence of flavin. (A) Step 7 protein (second heat treatment); (B) step 10 protein (second crystallization)! I % 1.0 P I I Protein concn. (rnglrnl) Fig.4. Dependence of the molecular weight of threonine dehydratase on protein concentration. The solvent was 0.05 M potassium phosphate buffer ph 7.6, containing 0.5 mm L-valine. All samples were centrifuged at rev./min for 16 and 24h at 5 C. Each molecular weights were calculated from high-speed sedimentation-equilibrium measurements by the method of Yphantis [lo] 0.i M triethanolamine-hc1 buffer over a ph range of 6.5 to 8.6. The reaction mixtures contained 16.7 mm L-threonine. The optimal ph for the reaction was about 8.2. This value is in agreement with earlier observations on partially purified enzyme [3,13]. Dependency of the Reaction Rate on the Concentration of Substrate The effect of substrate concentration on enzyme activity is shown in Fig. 6. The sigmoidal dependence is transformed to the classical Michaelis type in the presence of L-valine. An evaluation of these data according to Hill [I41 revealed n values of 2.16 and 0.98 for the interaction coefficients in the absence and presence of L-valine, respectively. At 3.3 mm L-t,hreonine the concentration of L-valine giving halfmaximal activation of the enzyme was 45 pm. Substrate Specificity In agreement with earlier observations on partially purified preparations [3-6,131, the purified enzyme catalyses the irreversible conversions of L-threonine into 2-oxobutyrate arid ammonia, and of L-serine into pyruvate and ammonia. However, the enzyme does not catalyse the deamination of L-homoserine. A comparison of the reaction rates with the three substrates in the presence of valine is given in Table 2. The apparent Michaelis constants were 7 mm for L-threonine, and 67 mm for L-serine.

5 Vol. 21, So. 1, 1971 T. KA4TSUNUMA, 8. ELSASSER, and H. HOLZER ar C m v Wavelength (nm) Fig. 5. Absorption spectrum of threonine dehydratase. Free pyrodoxal 5-phosphate was removed from the crystallized enzyme by gel-filtration on Sephadex G-75, previously equilibrated with 0.05 M phosphate buffer ph 7.6. The protein concentrations were 0.35 mg/ml (1) and 3.5 mg/ml (11); the light path was 1 cm Threonine (mm) Fig. 6. Dependence of the activity of threonine dehydratase on the concentration of L-threonine in the presence and absence of L-valine. Enzyme activity was measured as described in Methods with 0.5 pg protein (total volume = 3 ml, d = 1 cm). Concentration of L-valine 1 mm Table 2. K, and V of threonine dehydratase with threonine, serine and homoserine All assay solutions contained 10 mm L-valine Substrate Ii, V mm pmol x mg-' x mir3 L-Threonine L-Serine L-Homoserine - < 1.4 DISCUSSION A molecular weight of & 9500 was measured by high-speed sedimentation-equilibrium according to Yphantis [lo]. This value lies in the range of those found for threonine dehydratases from Bacillus subtilis ( [15]), Escherichia coli ( [Ill), and Xalnaonella typhimuriuna ( [12]). Whether the enzyme subunits exhibit cooperation similar to that observed with the bacterial enzymes, remains to be investigated. The purified enzyme is intrinsically unstable. It can, however, as previously described for crude preparations, be stabilized by addition of pyridoxal phosphate and L-valine or L-isoleucine. Sedimentation of the enzyme in sucrose gradients [16] is not influenced by L-threonine, L-valine, or L-isoleucine ; thus the state of aggregation of the enzyme is not affected by these compounds. The reaction velocity with pure enzyme, as also with crude and partially purified enzyme [6,13], is a sigmoid function of concentration of L-threonine. Addition of L-valine causes a transition to hyperbolic kinetics. This kinetic transition, as well as inhibition by L-isoleucine, is qualitatively and quantitatively similar to that observed in crude extracts. The properties of the purified enzyme may thus with some confidence be extrapolated to the enzyme in intact cells. We thank Dr E.-G. Afting and Miss H. Miiller for their generosity in determination of the molecular weight. Thanks are also due to Dr R. Wohlhueter for critical help with the manuscript. REFERENCES 1. Umbarger, H. E., Science (Washington), 123 (19) Umbarger, H. E., Cold Spring Harbor Symp. Quant. Biol. 26 (1961) Holzer, H., Boll, M., and Cennamo, C., Angew. Chem. 75 (1963) 894'and Angew. Chem. Int. Ed. 3 (1964) Cennamo, C., Boll, M.," and Holzer, H., Biochem (1964) Holzer. H.. Cennamo. C.. and Boll. M.. ' Biochem. Biophys. Re;. Commun. 14 (1964) Boll, M., and Holzer, H., Biochem (1965) Katsunuma, T., ElsaBer, S., and Holzer, H., Hoppe- Seyler's 2. Physiol. Chem. 352 (1971) Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. BioZ. Chem. 193 (1951) Schachman, H. K., Methods EnzymoZ. 4 (1957) Yphantis, D. A., Biochemistry, 3 (1964) Shizuta, Y., Nakazawa, A., Tokushige, M., and Hayaishi, O., J. Biol. Chem. 244 (1969) Zarlengo, M. H., Robinson, G. W., and Burns, R. O., J. Biol. Chem. 243 (1968) De Robichon-Szulmajster, H., and Magee, P. T., Eur. J. Biochem. 3 (1968) Brown, W. E. L., and Hill, A. V., Proc. Roy. Soc. (London) Ser. B, 94 ( ) Hatfield, G. W., and Umbarger, H. E., J. Bid. Chem. 245 (1970) Katsunuma, T., unpublished observations. T. Katsunuma, S. ElsaBer, and H. Holzer Biochemisches Institut der Universitat BRD-7800 Freiburg i. Br., Hermann-Herder-Strale 7 German Federal Republic