Branched Chain Amino Acid Aminotransferase of Pseudomonas

Agric. Biol. Chem., 41 (7), 1171 `1177, 1977 Branched Chain Amino Acid Aminotransferase of Pseudomonas Yuji KOIDE, Mamoru HONMA and Tokuji SHIMOMURA Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo, Japan Received December 20, 1976 sp. Branched chain amino acid aminotransferase was partially purified from Pseudomonas sp. by ammonium sulfate fractionation, aminohexyl-agarose and Bio-Gel A-0.5 m column chro matography. This enzyme showed different substrate specificity from those of other origins, namely lower reactivity for L-isoleucine and higher reactivity for L-methionine. Km values at ph 8.0 were calculated to be 0.3 mm for L-leucine, 0.3 mm for a-ketoglutarate, 1.1 mm for a-ketoisocaproate and 3.2 mm for L-glutamate. This enzyme was activated with ƒà-mercaptoethanol, and this activated enzyme had dif ferent kinetic properties from unactivated enzyme, namely, Km values at ph 8.0 were calculated to be 1.2 mm for L-leucine, 0.3 mm for a-ketoglutarate. Isocaproic acid which is the substrate analog of L-leucine was competitive inhibitor for pyridoxal form of unactivated and activated enzymes, and inhibitor constants were estimated to be 6 mm and 14 mm, respectively. In the previous paper,1) four L-alanine-aketo acid aminotransferases were separated from Pseudomonas sp. (a-aminoisobutyrate utilizing bacteria), and substrate specificity was examined for each enzyme. These re sults suggested that one of them would be branched chain amino acid-a-ketoglutarate aminotransferase (EC 2.6.1.42.). It has been known that this enzyme exists in organisms widely, especially animal en zyme has been studied in detail by Ichihara2 `6) and Taylor and Jenkins.7 `9) However, the enzymes of microorganism have not been purified and studied in detail except for Salmonella typhimurium,10) Pseudomonas aeru ginosa,l1) Neurospora crassal2) and Escherichia coli.13,14) Then, this paper presents the purification and some properties of the purified branched chain amino acid aminotransferase from Pseudomonas sp. MATERIALS AND METHODS Preparation of cell free extract. Cultivation of Pseudomonas sp. and preparation of cell free extract were described in the previous paper.1) Enzyme assay. Branched chain amino acid-aketoglutarate aminotransferase was assayed by the forward reaction with mixture containing 8 ƒêmoles of L-leucine, 4,umoles of a-ketoglutarate (a-kg), 40 ƒêmoles of potassium phosphate buffer (ph 8.0) 0.04ƒÊmole of pyridoxal 5'-phosphate and enzyme in the final volume of 0.4ml. The reaction was per formed at 30 Ž and terminated with 0.1 ml of 1.5 N HCl. Produced a-ketoisocaproate (a-kic) was measured by the method of Coleman.10) One unit of the enzyme activity represents the amount of enzyme forming I ƒêmole of a-kic per min at 30 Ž. Protein concentration was measured by micro-biuret method.15) For kinetic analysis, the reaction was carried out in 2.8 ml of mixture containing 280 moles of potassium phosphate buffer (ph 8.0), 0.28 ƒêmole of pyridoxal 5'-phosphate, varied concentrations of substrates and the enzyme at 30 Ž, and terminated by the addition of 0.2 ml of 3 N HCl. The determination of a-kic was made according to the method of Taylor and Jenkins16) except that 0.1 % dinitrophenylhydrazine solution was used. Amino acids formed in the reaction mixture were determined by the method of Tochikura.17 The reaction mixture of 0.4 ml contained 0.04 ƒêmole of pyridoxal 5'-phosphate and various amounts of L- amino and a-keto acids in 0.1 M potassium phosphate buffer (ph 8.0). The reaction was terminated with 0.1 ml of 1.5 N HCl, and 20 Id or 40 ƒêl aliquots were used to the assay. The amino acids were separated by paper chromatography using Toyo filter paper No. 50

1172 Y. KOIDE, M. HONMA and T. SHIMOMURA and the following solvent system: n-butanol-acetic acid-water (4-1-1) or phenol-water (3-1). For the separation of L-leucine and L-isoleucine 2-methylpro pan-2-ol-methyl ethyl ketone-28 % ammonia-water (5-3-1-1) was used.18) Preparation of aminohexyl-agarose. Aminohexyl - agarose was prepared by the method of Shaltiel.19) RESULTS Purification procedure All operations were performed at 0 `5 Ž in the presence of 0.01 % ƒà-mercaptoethanol. Step 1. Ammonium sulfate fractionation (30 `60%) was carried out according to the method previously described.1) Step 2. Dialyzed enzyme solution was applied to an aminohexyl-agarose column (3.0 ~9.0 cm) which was equilibrated with 0.01 M potassium phosphate buffer (ph 7.5), and eluted with KCl linear gradient in the range of 0 to 0.6 M. The elution pattern is shown in Fig. 1. Step 3. Active fractions were concen trated, and applied to a Bio-Gel A-0.5 m column, and eluted with 0.05M potassium phosphate buffer (ph 7.5). The results of partial purification are shown in Table I. By using aminohexyl-agarose, branched chain amino acid aminotransferase was completely separated from L-tryptophan-a-KIC aminotransferase20) which was not clearly separated with other chromatographic techniques; DEAE-Sephadex A-50, DEAE-cellulose or gel filtrations. At this point, although this finally prepared enzyme was not completely pure, it was used for the following experi ments. Substrate specificity Amino donor specificity with a-kg as amino acceptor is shown in Table II. This enzyme showed high activity on L-leucine, L-methionine and L-valine, and low activity on L-isoleucine. Amino acceptor specificity with several L-amino acids is shown in Table III. Although this enzyme catalyzed transamination from branched chain amino acids to related a-keto acid analogs, a-kg was the best amino ac ceptor. FIG. 1. Chromatography of Branched Chain Amino Acid Aminotransferase on Aminohexyl-agarose. Column size, 3 ~9 cm; buffer, 0.01 M potassium pho sphate (ph 7.5); elution, 0 `0.6 M KCl linear gradi ent; \, absorbance at 280 rim; \, L-leucine-a- KG transaminase activity; œ \ œ, L-tryptophan-a- KIC transaminase activity; ~ \ ~, KCI concentration. Michaelis constants of the enzyme In the forward reaction at ph 8.0, Kin values were calculated to be 0.3 mm for both L-leucine and a-kg (Fig. 2) with the method of Velick and Vavra.21) In the reverse reaction, higher concentra tion of a-kic caused substrate inhibition (Fig. 3). Because that the enzyme assay is TABLE I. PURIFICATION PROCEDURE OF BRANCHED CHAIN AMINO ACID AMINOTRANSFERASE

Branched Chain Amino Acid Aminotransferase of Pseudomonas sp. 1173 FIG. 2. Double Reciprocal Plot of Initial Velocities against L-Leucine Concentration at Series of Fixed Concentrations of a-kg. The experiments were carried out in 2.8 ml reaction mixture as described in the text. TABLE II. AMINO DONOR SPECIFICITY WITH a-kg The concentrations of amino donor and a-kg are 10 mm. FIG. 3. Substrate Inhibition of a-kic at the Con stant Concentration of L-Glutamate. The reaction mixture of 0.4 ml contained 0.04 Đmole of pyridoxal 5'-phosphate, I Đmole of L-glutamate and various amounts of a-kic in 0.1 M potassium phosphate buffer (ph 8.0). based on paper chromatography,17) it is difficult to calculate Km values of a-kic and L-glutamate at lower region of concentration. a-kic is the competitive inhibitor to pyri doxal form of the enzyme and inhibitor con stant of a-kic was obtained to be 3.8 mm by Dixon's plot (Fig. 4). To calculate Km value for L-glutamate, the data of Fig. 4 was re plotted to double reciprocal plot. Obtained apparent Km values for L-glutamate were plotted against a-kic concentration (Fig. 5). From intersections to horizontal and vartical axes, inhibitor constant for a-kic and Km value for L-glutamate were estimated to be 3.8 mm and 3.2 mm, respectively. Km value for a-kic was calculated from the equation, S=_??_22) Here, S is the concentration of a-kic which gives apparent maximal velocity TABLE III. AMINO ACCEPTOR AND DONOR SPECIFICITY Pyru, pyruvate; a-kb, a-ketobutyrate; a-kiv, a-ketoisovalerate; L-Abu, L-a-aminobutyrate.

1174 Y. KOIDE, M. HONMA and T. SHIMOMURA FIG. 4. Dixon's Plot of L-Glutamate-a-KIC Tran samination. The reaction mixture of 0.4 ml contained 0.04 µmole of pyridoxal 5'-phosphate and various amounts of L-glutamate and a-kic in 0.1 M potassium phosphate buffer (ph 8.0). FIG. 6. Effect of ƒà-mercaptoethanol on Enzyme Activity. The enzyme was treated with each concentration of ƒà-mercaptoethanol for 10 min ( \ ) or 20 min ( œ \ œ) at 30 Ž, and preincubated with pyridoxal 5'-phosphate for 10 min, then incubated with sub strates for 10 min. captoethanol (Fig. 6). In subsequent ex periments, the activated enzyme was prepared by the treatment with 0.1 M ƒà-mercaptoethanol in 0.1 M potassium phosphate buffer (ph 8.0) for 20 min at 30 Ž. Michaelis constants of 0.1 M ƒà-mercapto ethanol-activated enzyme FIG. 5. Estimation of Km Value for L-Glutamate and Inhibitor Constant for a-kic. In the forward reaction at ph 8.0, Km The data of Fig. 4 was replotted to double reciprocal plot and obtained apparent Km values for L-glutamate were plotted against a-kic concentration to calculate Km value for L-glutamate and Ki value for a-kic. in Fig. 3. Km value for a-kic (Ka) was calculated to be 1.1 mm, when S=2.0 mm and Ki=3.8 mm were substituted in the equation. Effect of ƒà-mercaptoethanol on enzyme activity In general, branched chain amino acid aminotransferase and leucine aminotrans ferase are activated with ƒà-mercaptoetha nol.4 `6,8) Importance of SH groups in active center of the enzyme was pointed out because that a small amount of p-chloromercuribenzoate caused inactivation.8,12) This en zyme from Psendomonas sp. was similarly activated 2-fold with above 50 mm ƒà-mer FIG. 7. Double Reciprocal Plot of Initial Velocities against L-Leucine Concentration at Series of Fixed Concentrations of a-kg in Activated Enzyme. The experiments were carried out under the conditions described in Fig. 5. The used enzyme was activated with 0.1 M ƒà-mercaptoethanol.

Branched Chain Amino Acid Aminotransferase of Pseudomonas sp. 1175 FIG. 8. Dixon's Plot of Isocaproic Acid Inhibition on L-Leucine-a-KG Transamination. The reaction mixture of 2.8 ml contained 0.28 ƒêmole of pyridoxal 5'-phosphate, 14 ƒêmoles of a-kg and various amounts of L-leucine and isocaproic acid in 0.1 M potassium phosphate buffer (ph 8.0). values were calculated to be 1.2 mm for L- leucine and 0.3 mm for a-kg (Fig. 7). In comparison with unactivated enzyme, ac tivated enzyme revealed greater Km value for L-leucine. This result suggests the participa tion of SH groups in the binding of L-leucine to enzyme. Inhibition of transamination by carboxylic acids It was reported that carboxylic acids as substrate analog are inhibitor of branched chain amino acid aminotransferase from pig heart. 9) Then, a series of mono- and di carboxylic acids were tested as inhibitor of transamination between L-leucine and a-kg. Both unactivated and activated enzymes were effectively inhibited by long chain mono carboxylic acids (Table IV). It is of interest that isocaproic acid which is substrate analog TABLE IV. INHIBITION BY CARBOXYLIC ACIDS The reaction mixture (2.8 ml) contained 5.6 µmoles of L-leucine and a-kg, and 56 ƒêmoles of carboxylic acid. of leucine is more effective inhibitor, but glutaric acid which is substrate analog of a-kg is less effective inhibitor. Inhibitor constants of isocaproic acid to pyridoxal form were measured as to unactivated and activated enzymes by the method of Velick and Vavra.21) It was found that isocaproic acid was a competitive inhibitor to L-leucine (Fig. 8), and the inhibitor constants were obtained to be 6 mm for unactivated enzyme and 14 mm for activated enzyme. DISCUSSION Branched chain amino acid aminotrans ferase was partially purified from Pseudomonas sp., and by using aminohexyl-agarose, another enzyme which catalyzes transamination be tween branched chain amino acids and a-keto analogs was completely separated as shown in Fig. 1. Partially purified branched chain amino acid aminotransferase showed extremely lower reactivity for L-isoleucine and higher reactivity for L-methionine. Generally, this enzyme represents about equal reactivity on L-leucine and L-isoleucine2 `7,10 `13) and low reactivity on L-methionine.6,7,11,12) At this point, the enzyme of Pseudomonas sp. was different from those of other origins. From the data of kinetics and substrate specificity, it could be considered that L-leucine-a-KG reaction pair is physiologically the most dominant.

1176 Y. KOIDE, M. HONMA and T. SHIMOMURA SCHEME I. Regulation of Branched Chain Amino Acid Pool by Aminotransferase System. Recently the authors purified L-tryptophana-KIC aminotransferase from the same or ganism that catalyzes transamination from only aliphatic or aromatic amino acids to a-keto analogs20) (Fig. 1). These two trans amination systems seem to maintain the branched chain amino acid pool in this or ganism as described in Scheme I. Similar transaminases have been reported in pea sprouts, valine-glutamate and valine-isoleucine aminotransferases. 23) Branched chain amino acid aminotrans ferase of Pseudomonas sp. was activated with Ĉ-mercaptoethanol, and this activated enzyme showed different kinetic properties from unactivated enzyme, such as increasing in Km value for L-leucine, Ki value for isocaproic acid and the maximum velocity. These kinetic properties were different from those of pig heart enzyme by Taylor and Jenkins.9) Pig heart enzyme was similarly activated with 0.1 M Ĉ-mercaptoethanol, however, this ac tivated enzyme showed decreasing Km value for L-leucine. To elucidate these differences, we considered the existence of S-S linkage in unactivated enzyme and SH groups in ac tivated enzyme from Pseudomonas sp. in the region of side-chain binding site for L- leucine. REFERENCES 1) Y. Koide, M. Honma and T. Shimomura, Agric. Biol. Chem., 41, 781 (1977). 2) A. Ichihara and E. Koyama, J. Biochem., 59,160 (1966). 3) A. Ichihara, H. Takahashi, K. Aki and A. Shirai, Biochem. Biophys. Res. Commun., 26, 674 (1967). 4) K. Aki, K. Ogawa, A. Shirai and A. Ichihara, J. Biochem., 62, 610 (1967). 5) K. Aki, A. Yokojima and A. Ichihara, ibid., 65, 539 (1969). 6) K. Ogawa, A. Yokojima and A. Ichihara, ibid., 68, 901 (1970). 7) R. T. Taylor and W. T. Jenkins, J. Biol. Chem., 241, 4396 (1966). 8) R. T. Taylor and W. T. Jenkins, ibid., 241, 4406 (1966). 9) R. T. Taylor and W. T. Jenkins, ibid., 245, 4880 (1970). 10) M. S. Coleman and F. B. Armstrong, Biochim. Biophys. Acta, 227, 56 (1971). 11) J. E. Norton and J. R. Sokatch, ibid., 206, 261 (1970). 12) M. Collins and R. P. Wagner, Arch. Biochem. Biophys., 155, 184 (1973). 13) D. Rudman and A. Meister, J. Biol. Chem., 200, 591 (1953). 14) R. Raunio, Acta Chem. Scand., 22,2733 (1968). 15) R. F. Itzhaki and D. M. Gill, Annal. Biochem., 9, 401 (1964). 16) R. T. Taylor and W. T. Jenkins, J. Biol. Chem., 241, 4391 (1966). 17) T. Tochikura, T. Tachiki, K. Nakahama, A.

Branched Chain Amino Acid Aminotransferase of Pseudomonas sp. 1177 Baich and V. H. Cheldelin, Agric. Biol. Chem., 37, 1611 (1973). 18) J. G. Heathcote, Biochem. J., 97, 15 p (1965). 19) S. Shaltiel and Zui Er-EL, Proc. Natl. Acad. Sci. U.S.A., 70, 778 (1973). 20) Y. Koide, M. Honma and T. Shimomura, in pre paration. 21) S. F. Velick and J. Vavra, J. Biol. Chem., 237, 2109 (1962). 22) W. W. Cleland, "The Enzymes," 3rd Ed. Vol. II, ed. by P. D. Boyer, Academic Press Inc., New York, 1970, p. 35. 23) Z. S. Kagan and A. S. Dronov, Dokl. Akad. Nauk. SSSR., 179, 1236 (1968).