J. Biochem. 85, 901-906 (1979)

Purification and Properties of Pig Liver Kynureninase Katsuyuki TANIZAWA and Kenji SODA Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611 Received for publication, October 2, 1978

Kynureninase [L-kynurenine hydrolase, EC 3.7.1.3] was purified from pig liver by a procedure including DEAE-cellulose chromatography, hydroxyapatite chromatography, ammonium sulfate fractionation, DEAE-Bio Gel chromatography, Sephacryl S-200 gel filtration, kynurenine-Sepharose affinity chromatography, and Sephadex G-200 gel filtration. The enzyme was found to be homogeneous by the criterion of disc-gel electrophoresis. The enzyme has a molecular weight of about 100,000 and exhibits absorption maxima at 280 and 420 nm. The optimum pH and the isoelectric point of the enzyme are 8.5 and 5.0, respectively. The Michaelis constants were determined to be as follows: L-kynurenine, 7 . 7 X 1 0 ~ 4 M ; L-3-hydroxykynurenine, 1.3X1O~ 6 M; and pyridoxal 5'-phosphate, 1.8X10" 9 M.

L-3-Hydroxykynurenine

is hydrolyzed more rapidly than L-kynurenine; the liver enzyme can be regarded as a 3-hydroxykynureninase.

Kynureninase [L-kynurenine hydrolase, EC 3.7.1.3] was discovered in mammalian liver by Kotake and Nakayama (7), and partially purified by Wiss (2,5). Braunstein et al. (4) demonstrated a decrease of the kynureninase activity on pyridoxine deficiency, showing that the enzyme requires pyridoxal 5'phosphate (pyridoxal-P) as a coenzyme. Several otheT workers have studied the effect of pyridoxine deficiency on tryptophan metabolism, particularly on the kynureninase activity (5-7). All mammalian livers tested, e.g. mouse, guinea pig, dog, and human livers, contain high kynureninase activity, while the kidneys have much lower activity (8), and the activity of spleen, lung, brain, heart, and muscle is negligible (9). The livers of other vertebrates, i.e. birds, reptiles, amphibia, and fishes, also show kynureninase activity (JO), suggesting that the enzyme is indispensable in animal livers. Abbreviation: pyridoxal-P, pyridoxal 5'-phosphate. Vol. 85, No. 4, 1979

901

The mammalian kynureninase has not been purified sufficiently highly to permit characterization, although homogeneous preparations have been obtained from bacterial cells (11) and fungal mycelia (12). The present paper describes the purification of pig liver kynureninase to homogeneity and its properties. EXPERIMENTAL PROCEDURE Materials—Fresh pig livers were obtained from the Kyoto abattoir. Hydroxyapatite was prepared by the method of Tiselius et al. (13). DEAE-Bio Gel A and Sephacryl S-200 were purchased from Bio-Rad Laboratories and Pharmacia, respectively. L-Kynurenine was synthesized from L-tryptophan by the method of Warnell and Berg (14), and N'formyl-L-kynurenine was synthesized from Lkynurenine according to the method of Dalgliesh (15). L-3-Hydroxykynurenine was obtained from

PIG LIVER KYNURENINASE

903

Step 4: The precipitate was washed with 500 ml of 10 mM buffer (pH 7.2) containing 60% ammonium sulfate, and dissolved in 200 ml of the buffer containing 40 % ammonium sulfate. Insoluble protein was removed by centrifugation. The enzyme in the supernatant solution was precipitated by addition of ammonium sulfate (80% saturation) and dissolved in a small volume of 10mM buffer (pH 7.2) followed by dialysis against 100 volumes of 5 mM buffer (pH 7.2). Step 5: The dialyzed enzyme was applied to a DEAE-Bio Gel column (2 x 30 cm) equilibrated with 5 mM buffer (pH 7.2), and eluted with a linear gradient formed from 1,500 ml of 5 mM buffer (pH 7.2) and 1,500 ml of 5 mM buffer containing 0.2 M KC1. The enzyme was eluted with buffer containing about 0.12 M K G . The active fractions were pooled and concentrated by ultrafiltration through a Diaflo membrane (Amicon). Step 6: The enzyme solution was applied to a Sephacryl S-200 column (2.5x120 cm) equilibrated with 10 mM buffer (pH 7.2) and eluted with the same buffer. The active fractions were collected and concentrated by ultrafiltration. Step 7: The enzyme solution was applied to a column (1.2x15 cm) of L-kynurenine-bound Sepharose 4B previously equilibrated with 50 mM buffer (pH 8.0). The column was washed with 0.1 M buffer (pH 8.0), and the enzyme was eluted with 0.1 M buffer (pH 8.0) supplemented with 0.3 M KC1 (Fig. 1). The active fractions were concentrated by addition of ammonium sulfate (70% saturation). The precipitate was dissolved in a small volume of 50 mM buffer (pH 7.2).

Step 8: The enzyme was placed on a Sephadex G-200 column (2x110 cm) equilibrated with 50 mM buffer (pH 7.2) and eluted with the same buffer. The active fractions were combined and concentrated by ultrafiltration. The enzyme (approximately 2.5 mg/ml) was stored at —20°C in 50 mM buffer (pH 7.2). A summary of the purification procedure is shown in Table I. The enzyme was purified about 2,000-fold with an overall yield of about 9%. Homogeneity and Molecular Weight—The purified enzyme showed a single protein band upon disc-gel electrophoresis (Fig. 2). To determine whether the band corresponded to the enzyme activity, a gel was run in the normal way, and a thin vertical slice of the gel was stained with Coomassie brilliant blue then destained with 10% acetic acid solution in 10% methanol. The section of the unstained gel corresponding to the protein band was cut out and crushed in a small volume

Fig. 2. Disc-gel electrophoresis of pig liver kynureninase. A sample of the purified enzyme preparation (35 fig) was electrophorescd under the conditions of Davis (18).

TABLE I. Purification of pig liver kynureninase.1 Step 1. 2. 3. 4. 5. 6. 7. 8.

Crude extract DEAE-cellulose chromatography Hydroxyapatite chromatography Ammonium sulfate fractionation DEAE-Bio Gel chromatography Sephacryl S-200 gel filtration Kyn-Sepharose 4B chromatography6 Sephadex G-200 gel filtration

Total protein (mg) 167,300 20,513 5,322 1,654 873 487 23.7 7.04

Total units 54.1 24.6 23.0 16.7 15.5 14.0 13.3 4.65

Specific activity

Yield

0.000323 0.00120 0.00432 0.0101 0.0177 0.0288 0.563 0.661

L-Kynurenine was used as a substrate. >> L-Kynurenine-bound Sepharose 4B affinity chromatography. Vol. 85, No. 4, 1979

100 45 43 31 29 26 25

9

904

K. TANIZAWA and K. SODA

of 10 mM potassium phosphate buffer (pH 7.2) containing 20 ^M pyridoxal-P. After elution for 20 min at 4°C, the gel was removed by centrifugation, and the supernatant solution was found to contain kynureninase activity. The molecular weight of the enzyme was estimated from the elution volume on Sephadex G-200 column chromatography (21). Protein solution (0.5 ml) was applied to a Sephadex G-200 column (1.5x120 cm) equilibrated with lOmM potassium phosphate buffer (pH 7.2) containing 0.1 M KC1, and eluted with the same buffer. The column was standardized with egg albumin (molecular weight, 43,000), bovine serum albumin (68,000), bacterial kynureninase (91,000) (22), bovine heart lactate dehydrogenase (140,000), and bovine liver catalase (240,000). An average value of approximately 100,000 was obtained.

T100

Isoelectric Point—The isoelectric point of the enzyme was determined to be 5.0 by isoelectric focusing on polyacrylamide gels, as shown in Fig. 3. Absorption Spectrum—The enzyme exhibited absorption maxima at 280 and 420 nm (Fig. 4), showing that it contains pyridoxal-P as a prosthetic group. The absorbance ratio at 280 and 420 nm was about 100 : 5. The apoenzyme was prepared by incubation of the holoenzyme with 5 mM phenylhydrazine in 0.1 M potassium phosphate buffer (pH 7.2) at 25°C for 30 min followed by gel nitration on Sephadex G-25 equilibrated with 10 mM potassium phosphate buffer (pH 7.2). It showed no absorption peak at 420 nm (Fig. 4). Effect of pH—The enzyme showed the highest activity at pH 8.5 in 50 mM Tris-HCl buffer. At the pH optimum, similar activities were found in 50 mM sodium pyrophosphate-HCl and glycineKC1-KOH buffers. Substrate Specificity and Kinetics—When the initial velocity of the enzyme reaction was measured TABLE II. Effect of inhibitors on the pig liver kynureninase activity. The enzyme was incubated with the compounds listed (1 mM except p-chloromercuribenzoate (0.5 mM)) at 25°C for 10 min. The reaction was started by addition of L-kynurenine.

G«l

10 Slice

15 20 Number

25

Fig. 3. Isoelectric point determination. The purified pig liver kynureninase (60 /ig) was electrofocused under the conditions described in the text.

300

BO Wavatongth

tOO (run)

Fig. 4. Absorption spectra of pig liver kynureninase. Curve A, holoenzyme in 10 mM potassium phosphate buffer (pH 7.4); Curve B, apoenzyme prepared as described in the text, in 50 mM potassium phosphate buffer (pH 7.4).

Compounds

Rel. Act.

None Phenylhydrazine Hydroxylamtne Semicarbazide D-Cycloserine Potassium cyanide L-Penicillamine D-Penicillamine HgCl, />CMB» JV-Ethylmaleimide Iodoacetate EDTA* MgCl, MnCl, CaClt

100 0 0 34 85 51

58 65 5 18 43

63 100 102

100 101

»pCMB, ^-chloromercuribenzoate; EDTA, ethylenediaminetetraacetic acid. / . Biochem.

PIG LIVER KYNURENINASE spectrophotometrically (12) in a reaction mixture containing 0.195 mM L-kynurenine, 0.2 mM L-3hydroxykynurenine, or 1 nut iV'-formyl-L-kynurenine, the relative activities were found to be 100, 1,038, and 5.6, respectively. The D-enantiomers were not substrates. The Michaelis constant was determined from a double-reciprocal plot of the relationship between the reaction velocity and the substrate or cofactor concentration. The apparent Km values were as follows: L-kynurenine, 7.7x

905

ninase: one of them is formed inducibly by tryptophan (and also by kynurenine) and participates in the catabolism of tryptophan (kynureninase or inducible kynureninase), and the other is a constitutive enzyme and functions in the biosynthesis of NAD from tryptophan (3-hydroxykynureninase or constitutive kynureninase) (29, 30). Both Ps. fluorescens IFO 3925 and N. crassa JFO 6068 produce the inducible kynureninase (11, 12). Shetty and Gaertner (31) observed a constitutive 10"* M; L-3-hydroxykynurenine, 1.3X10~ 5 M; and kynureninase in Saccharomyces cerevisiae. They pyridoxal-P, 1 . 8 X 1 0 " ' M . The Km value for the suggested on the basis of kinetic analysis that the cofactor was obtained by assaying the activity after yeast enzyme functions biosynthetically in the same incubation of the apoenzyme with various amounts way as the constitutive enzyme of N. crassa (Taniof pyridoxal-P in 10 mM potassium phosphate buffer zawa, K. and Soda, K., manuscript in preparation). (pH 7.5) at 25°C for 30 min. The activity of liver kynureninase is not affected Inhibitors—Various compounds were investi- by the intraperitoneal administration of kynurenine gated for their inhibitory effects on the enzyme or tryptophan (5), although tryptophan dioxygenase activity (Table II). The enzyme was inhibited is induced by adrenal cortical hormones or tryptomost strongly by hydroxylamine and phenyl- phan (32-34). hydrazine, both of which are typical inhibitors of The present studies show that the liver kynurepyridoxal-P enzymes. Semicarbazide, L- and D- ninase resembles the inducible enzymes of Ps. penicillamine, potassium cyanide, and D-cyclo- fluorescens (22) and N. crassa (12) in several reserine were also inhibitory. Thiol reagents e.g. spects, e.g. molecular weight and optimum pH. HgCl, and />-chloromercuribenzoate, significantly However, the liver enzyme hydrolyzes 3-hydroxyinhibited the enzyme. These inhibitory effects of kynurenine about 10 times more rapidly than carbonyl and thiol reagents on the enzyme are kynurenine, and the affinity for 3-hydroxykynuresimilar to those on bacterial (22) and fungal nine is approximately 60 times higher than that kynureninase (12). Ethylenediaminetetraacetic for kynurenine. Thus, the liver kynureninase can acid and divalent cations such as Mg1+, Mn2+, be regarded as a 3-hydroxykynureninase, and and Ca1+ had no effect on the activity. probably functions in the tryptophan-NAD pathway as reported for the constitutive enzymes of N. crassa and S. cerevisiae. Ueda et al. (35) DISCUSSION reported that 3-hydroxyanthranilic acid is formed Kynureninase functions as a key enzyme of the from anthranilic acid, a product of kynurenine aromatic and NAD pathways in tryptophan hydrolysis, by a hydroxylating enzyme in micrometabolism, and catalyzes a unique reaction, the somes of rat liver, and suggested that the hydroxylhydrolytic /9, y cleavage of aryl-substituted f-keto- ation provides an alternative route of NAD a-amino acids. Several attempts have been made biosynthesis from tryptophan (tryptophan •» to purify kynureninase from mammalian sources, kynurenine —• anthranilic acid —• 3-hydroxyanthrawith little success (5, 23-28). We have now nilic acid - m NAD). If this is also the case for purified the enzyme to homogeneity from pig liver. the pig liver system, kynurenine must be the The purified enzyme has the highest specific activity preferred substrate of kynureninase. However, the among the preparations obtained so far. The kinetic analysis of the liver kynureninase described preparation of homogeneous mammalian kynure- above shows that 3-hydroxykynurenine is the best ninase was suitable for comparative studies of it and probably the physiological substrate of the and the enzymes of Pseudomonasfluorescens(=Ps. enzyme. Therefore, kynurenine is probably converted into 3-hydroxykynurenine before its hydrolmarginalis) (11) and Neurospora crassa (12). ytic cleavage by kynureninase, and the main Recently, ample evidence has been obtained biosynthetic pathway of NAD from tryptophan for the occurrence of two distinct types of kynureVol. 85, No. 4, 1979

IC TANIZAWA and K. SODA

906

may be as follows; tryptophan-H kynurenine —* 3-hydroxykynurenine —» 3-hydroxyanthranilic acid •mNAD. We thank Dr. T. Yamamoto and Dr. H. Yamada for valuable advice and discussions. REFERENCES 1. Kotake, Y. & Nakayama, T. (1941) Z. Physiol. Chem. 270, 76-82 2. Wiss, O. (1949) Helv. Chim. Ada 32, 1694-1698 3. Wiss, O. (1952) Z. Naturforsch. 76, 133-136 4. Braunstein, A.E., Goryachenkova, E.V., & Paskhina, T.S. (1949) Biokhimiya 14, 163-179 5. Knox, W.E. (1953) Biochem. J. 53, 379-385 6. Dalgliesh, C.E., Knox, W.E., & Neuberger, A. (1951) Nature 168, 20-22 7. Dalgliesh, C.E. (1952) Biochem. J. 52, 3-14 8. Knox, W.E. (1955) in Methods in Enzymology Vol. 2, Preparation and Assay of Enzymes (Colowick, S.P. & Kaplan, N.O., eds.) pp. 249-253, Academic Press, New York 9. Machill, G. (1972) Ada Biol. Med. Cer. 29, 179-183 10. Gaertner, F.H. & Shetty, A.S. (1977) Biochim. Biophys. Ada 482, 453-460 11. Moriguchi, M., Yamamoto, T., & Soda, K. (1971) Biochem. Biophys. Res. Commun. 44, 752-757 12. Tanizawa, K , Yamamoto, T., & Soda, K. (1976) FEBS Lett. 70, 235-238 13. Tiselius, A., Hjerten, S., & Levin, O. (1956) Arch. Biochem. Biophys. 65, 132-155 14. Warnell, J.L. & Berg, C.P. (1954) / . Amer. Chem. Soc. 76, 1708-1709 15. Dalgliesh, C.E. (1952)/. Chem. Soc. 137-141 16. Schlitt, S.C., Lester, G., & Russel, P.J. (1974) / . Baderiol. 117, 1117-1120

17. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, RJ. (1951) /. Biol. Chem. 193, 265-275 18. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121,404-427 19. Wrigley, C.W. (1968) /. Chromatogr. 36, 362-365 20. Wrigley, C.W. (1971) in Methods in Enzymology Vol. 22, Enzyme Purification and Related Techniques (Jakoby, W.B., ed.) pp. 559-564, Academic Press, New York 21. Andrews, P. (1964) Biochem. J. 91, 222-233 22. Moriguchi, M., Yamamoto, T., & Soda, K. (1973) Biochemistry 12, 2969-2974 23. Wiss, O. (1953) Z. Physiol. Chem. 293, 106-121 24. Wiss, O. & Weber, F. (1956) Z. Physiol. Chem. 304, 232-240 25. Ogasawara, N., Hagino, Y., & Kotake, Y. (1962) / . Biochem. 52, 162-166 26. Hagino, Y. (1964) Nagoya J. Med. Sci. 26, 221-227 27. McDermott, C.E., Casciano, D.A., & Gaertner, F.H. (1973) Biochem. Biophys. Res. Commun. 51, 813-818 28. De Antoni, A., Costa, C , & Allegri, G. (1975) Ada Vitamin. Enzymol. (Milano) 29, 339-343 29. Gaertner, F.H., Cole, K.W., & Welch, G.R. (1971) J. Baderiol. 108, 902-909 30. Shetty, A.S. & Gaertner, F.H. (1975) J. Baderiol. 122, 235-244 31. Shetty, A.S. & Gaertner, F.H. (1973) / . Baderiol. 113, 1127-1133 32. Knox, W.E. (1951) Br. J. Exp. Pathol. 32, 462^169 33. Madras, B.K & Sourkes, T.L. (1968) Arch. Biochem. Biophys. 125, 829-836 34. Young, S.N. & Sourkes, T.L. (1968) J. Biol. Chem. 250, 5009-5014 35. Ueda, T., Ohtsuka, E., Gohda, K., & Kotake, Y. (1978) in The Proceeding of the 25th Regular Meeting of the Kinki Branch of the Japanese Biochemical Society (Kobe) (in Japanese) p. 6

/ . Biochem.