Transaminase of Branched Chain Amino Acids

Transcription

1 The Journal of Biochemistry, Vol. 59, No. 2, 1966 Transaminase of Branched Chain Amino Acids I. Branched Chain Amino Acids-a-Ketoglutarate Transaminase By AKIRA ICHIHARA* and Eizo KOYAMA (From the Department of Biochemistry, Dental School, Osaka University, Osaka) (Received for publication, October, 7, 1965) It has been established that the first reaction in the metabolism of the majority of amino acids is transamination and many transaminases have been reported. Branched chain amino acids (e.g., valine, leucine, and isoleucine) are very similar in structure and are essential for animal nutrition. It is known that they have a competitive effect on each other in nutrition (1, 2) and that valine is glycogenic, while leucine is ketogenic and isoleucine is both (3 5). In spite of their physiological importance, the mechanism of their transamination in animals is not fully understood. There are several reports showing that they are transaminated (6 9), but for previous studies the enzyme preparations used were crude homogenates of various tissues, and branched chain amino acids were among the other amino acids of which the transamination was studied. Therefore, it is not known whether some transaminase with broad substrate specificity acts on branched chain amino acids as well as other amino acids, or whether former have their own specific transaminase and if so, whether this enzyme is common to them all or whether each branched chain amino acid has its own specific transaminase. Studies on transaminases for branched chain amino acids in micro-organisms have suggested that they do not have a specific enzyme, but that their transamination is catalyzed by an enzyme which also transaminates a number of other amino acids (10 13). This report is on the distribution of transaminase activities of the branched chain amino acids in various tissues of rats and also reports the partial purification and properties of a branched chain amino acid :2-oxoglutarate aminotransferase [EC 2.6.1] from hog heart. It is concluded that this enzyme is specific for branched chain amino acids and that these compounds are all transaminated by the same enzyme. Preliminary work on this subject has been reported (14). EXPERIMENTAL Assay Method The incubation mixture contained (in ^moles) : sodium pyrophosphate buffer (ph 8.6) 50, L-amino acid 10, ar-ketoglutarate 10, pyridoxal phosphate 0.1 and enzyme in a total volume of 1.5 ml. This mixture was preincubated at 37 C for 5 minutes, and then the reaction was started by addition of either amino acid or a-ketoglutarate. After 10 minutes the reaction was terminated by addition of trichloroacetic acid at a final concentration of 5 per cent. The amount of keto acid formed was determined as its hydrazone by a modification of the method of W a d a and Snell (15). The acidified reaction mixture was centrifuged if necessary and then the clear solution was transferred to a test tube with a glass stopper and preincubated for 5 minutes at 25 C C. Then 2 ml. of 0.5 per cent 2,4-dinitrophenylhydrazine in 2 N HC1 was added and the mixture was incubated for 5 minutes. Then 5 ml. of toluene was added and the mixture was shaken vigorously for 2 minutes. The water layer was removed through a capillary pipette * Present address: The Institute for Enzyme and discarded. Five ml. of 0.5 N HC1 was added to Research, School of Medicine, Tokushima University, the toluene layer and the mixture was shaken for Tokushima. Requests for reprints should be sent to 1 minute. It was then centrifuged to remove the this address. hydrazone of a-ketoglutarate and 2 ml. of the toluene 160

2 Branched Chain Amino Acids Transaminase. I 161 layer was pipetted out and mixed well with 2 ml. of 10 per cent sodium carbonate solution in another test tube. One ml. of the carbonate layer was taken out and mixed with lml. of 1.5A^NaOH. The optical density of this solution was measured at 440 mit. The hydrazones of the keto acids of the three branched chain amino acids showed similar absorption maxima at 440 m/j and these had similar extinction coefficients. Activity was expressed as m//mole of keto acid formed per 10 minutes. The reverse reaction was studied using the reaction mixture described above, except that L-glutamate and the keto acids of the branched chain amino acids were added instead of a-ketoglutarate and the branched chain amino acids. Hydrazone formation was carried out as described above until the stage of extraction witli toluene, except that the hydrazine was incubated with the mixture for 25 minutes. Then the toluene layer was transferred to another test tube with a glass stopper, the water layer was extracted again with 5 ml. of toluene and this extract was combined with the first toluene layer and washed with 5 ml. of 0.5 N HC1. The HC1 layer was then combined with the original water layer and extracted Organ Liver Heart muscle Skeletal muscle Kidney TABLE I Enzyme Distribution in Various Organs of the Rat Substrate with 5 ml. of ethyl acetate. Then 2 ml. of the ethyl acetate layer was pipetted out and mixed with 2 ml. of 10 per cent sodium carbonate solution. After vigorous shaking of the mixture, 1 ml. of the carbonate layer was mixed with lml. of 1.5AT sodium hydroxide solution and the optical density of the mixture was measured at 440 m^. The extinction coefficient was the same as that of the hydrazones of a-keto-monocarboxylates. When L-leucine-l-C u (16,375 c.p.m//zmole) was used as substrate, the hydrazone of ar-ketoisocaproate- 1-C' 4 was isolated as described above, and the toluene layer, after centrifugation, was transferred to a planchet and dried. The radioactivity was measured in a gas flow counter. Protein was measured by the Biuret reaction*. The keto acids of each of the three branched chain amino acids (a-ketoisovaleric, a-ketoisocaproic and D-a-keto-j3-methylvaleric acids) were prepared from the respective amino acids by the method of Meister (16). Aspartate transaminase [EC ] and alanine transaminase [EC ] purified from hog heart were generous gifts from Dr. H. Wad a of Osaka University. Activity per mg. protein (mfiraolc/lo min.) per g. wet tissue ,140 14,490 10, ,367 1,960 14,500 16,000 18,500 Rat organs were homogenized with 5 ml. of potassium phosphate buffer (ph 7.4) in a Potter-Elvehjem type homogenizer and the homogenates were centrifuged at 5,000xg for 15 minutes. The supernatant was incubated as described in " EXPERIMENTAL ", except that potassium phosphate buffer (ph 7.4) was used instead of pyrophosphate buffer. * Pardee, A.B., personal communication.

3 162 A. ICHIHARA and E. KOYAMA RESULTS Distribution of Transaminase Activity in Various Organs of the Rat The distribution of the activity of the enzyme which transaminates valine, leucine and isoleucine with a-ketoglutarate in various organs of the rat was investigated. Table I indicates that heart muscle and kidney showed similar activities and these were the highest, followed by skeletal muscle, while liver showed negligible activity. This order of activities is comparable with the results of former workers (7), except for the liver activity which in the present work was about one tenth of that reported in the literature. was found to be the best substrate, followed by isoleucine in heart and skeletal muscle, whereas in liver and kidney isoleucine was attacked more rapidly than leucine. Moreover, in all these tissues valine was attacked slowest of the three amino acids. These differences of substrate specificity were very different from those reported previously, in which the activities with the three amino Fraction Homogenate Mitochondria Microsomes Supernatant TABLE II Inlracellular Distribution of the Enzyme in Rat Heart Muscle Substrate Activity (m^mole/10 min.) (per g. original tissue) 4,000 14,000 9,800 1,400 4,000 2, ,400 7,200 4,400 Rat heart muscle was homogenized and fractionated according to the method of Hogeboom (/7). Incubation was carried out as described in Table I. acids were almost the same (6 8). This difference may be due to the inaccuracy of previous assays in which activities were measured with crude homogenates, incubating for one hour and estimating the product by paper chromatography or glutamate decarboxylase [EG ]. No other organs were examined, because Awapara and Seale reported that they had less activity than those of the organs used in the present work. However, it is interesting that there were relatively high activities in the prostate and testis (7). Table II shows the distribution of transaminase activity in subcellular fractions of rat heart muscle. It was found that the soluble fraction contained higher activity with all three substrates than the mitochondrial fraction, though the activity in the latter was also significant. Rowsell (8) reported that the activity was distributed in both the soluble and the particulate fractions of liver and kidney and it is known that aspartate transaminase and alanine transaminase are localized in both fractions (18 20). Purification of the Enzyme from Hog Heart Since preliminary work had showed that with «-ketoglutarate heart muscle has the highest transaminase activity for branched chain amino acids, hog heart was used as a source of the enzyme during this work. A sample of 430 g. of hog heart was homogenized three times with 830 ml. of 0.01 M potassium phosphate buffer (ph 8.0) in a Waring blendor and the homogenate was centrifuged at 10,000 Xg for 15 minutes. The resulting supernatant [Crude extract] was fractionated with solid ammonium sulfate and the precipitate formed between 35 and 70 per cent saturation was dissolved in 0.01 M potassium phosphate buffer (ph 8.0) containing 0.01 M mercaptoethanol and pyridoxal phosphate at a concentration of 3 fj%. per ml. and dialyzed against the same buffer. This buffer was found to stabilize the enzyme, and the same additions were made to all the buffer used in purification of the enzyme. The dialyzed enzyme [(NH^SOi-I] was applied to a DEAE-cellulose column which had been equilibrated with the buffer described above, and was eluted with the same buffer containing a concentration gradient of KC1 between

4 Branched Chain Amino Acids Transaminase. I 163 TABLE III Purification of the Enzyme from Hog Heart Muscle Crude extract (NH 4 ) 2 SO 4 -I DEAE-I (NH 4 ) 2 SO 4-1I DEAE-II (NH 4 ) 2 SO 4 -III Total volume (ml.) 2, , Total protein (mg-) 27, 360 9,570 4, Specific activity for leucine (^mole/10 min./mg. protein) ) Relative activities were calculated taking the activity with leucine as 1. ph of the buffer Mercaptoethanol Addition TABLE IV Stability of the Enzyme Pyridoxal phosphate a-ketoglutarate Yield (%) Relative activity" for Stability" for 8.0 1) Stability of the enzyme activity is expressed as a percentage of that of fresh enzyme with leucine. Enzyme was aged at 4 C for 39 hours in the various conditions described above. Buffer used was potassium phosphate at a concentration 0.01 M, except at ph 8.9 sodium pyrophosphate was added. The concentrations of other components added were; mercaptoethanol 0.01 M, pyridoxal phosphate 5/*g./ml., and o-ketoglutarate 2.5 /zmoles/ml., respectively. 0 and 0.2 M. The active fraction [DEAE-1] was collected and fractionated with ammonium sulfate. The precipitate formed between 40 and 60 per cent saturation was dissolved in, and dialyzed against the same buffer. After dialysis, the preparation was centrifuged and the clear supernatant [(NH 4 ) 2 SO 4-1I] was rechromatographed on DEAE-cellulose in the same way as previously. The active fraction [DEAE-II] was subjected to a third ammonium sulfate fractionation and the precipitate formed between 40 and 55 per cent saturation was dissolved in, and dialyzed against the same buffer [(NH 4 ) 2 SO4-III]. This material was 1. 1 used as the enzyme preparation. Typical results of the purification procedure are shown in Table III. The specific activity for leucine was about 60 fold that of the original crude extract and the yield was 10 per cent. During purification the ratio of the activities for the three branched chain amino acids remained constant. It was found that the activity ratio with leucine to that with isoleucine was somewhat different in rat heart and hog heart and that leucine was attacked preferentially in rat heart, whereas in hog heart isoleucine was the best substrate. The reason for this is unknown

5 164 A. ICHIHARA and E. KOYAMA Properties of the Enzyme The enzyme was rather unstable at ph 8 unless pyridoxal phosphate and mercaptoethanol were added, but the further addition of a-ketoglutarate did not increase its stability (Table IV). The enzyme was inactivated by heat, as shown in Fig. 1, and addition of the substrate did not prevent this heat inactivation. It is interesting that the activity towards each substrate decreased at the same rate. This, together with the fact that the activity ratios for each substrate were constant during the purification, strongly suggests that a single enzyme transaminates these three substrates. For enzyme activity one of the three branched chain amino acids, a-ketoglutarate and pyridoxal phosphate were essential and the K m values for each component were: valine l.lxlfj- 2 M, leucine 3.8xlO- 3 M, isoleucine 3.8xlO" 3 M (Fig. 2), a-ketoglutarate 6.3xlO~ 4 Af (Fig. 3) and pyridoxal phosphate \ \ n i l l? TEMPERATURE Ct) FIG. 1. Heat stability of the transaminase activities with the three substrates. Enzyme solution containing 10 //moles of potassium phosphate buffer (ph 8.0), 10 //moles of mercaptoethanol and 8 fig. of pyridoxal phosphate in one ml. was heated as described in the figure for 5 minutes. Enzyme activities were expressed with respective substrates as percentages of those with the unheated preparation. : valine; O : leucine; A : isoleucine. O.I O.z 0.3 1/5 FIG. 2. Effect of valine, leucine and isoleucine concentrations on enzyme activity. : valine; O : leucine; A : isoleucine. O.I 0.2 Q I /a-ketoglutaratefxio-"/*/] FIG. 3. Effect of a-ketoglutarate concentration on enzyme activity.

6 Branched Chain Amino Acids Transaminase. I 165' FIG. 4. Effect of pyridoxal phosphate concentration on enzyme activity. FIG. 5. ph optima of enzyme activities for the three substrates. : valine, O : leucine; A : isoleucine. 6.7 x 10~ 5 M (Fig. 4). For determination of the K m for pyridoxal phosphate it was necessary to dialyze the enzyme several times against the buffer described above, but containing 1x10 s M hydroxylamine instead of pyridoxal phosphate and finally against buffer containing only mercaptoethanol. In this way the pyridoxal phosphate was dissociated completely from the enzyme. The optimal ph values for activity with the three substrates were all about ph 8.6 (Fig. 5). Tris buffer reduced the activity by about one half. />-Chloromercuribenzoate completely inhibited enzyme activity at a concentration of 1 x 10~ 4 M. Addition of ATP or AMP at a concentration of 5xlO~ 4 M did not affect the activity. Substrate Specificity The transaminase showed high substrate specificity for the branched chain amino acids, but it also showed some activity with norvaline and norleucine (Table V). The amino acids with which it had no activity were alanine, aspartic, a-amino and?--aminobutyric, -aminocaproic acids, ornithine, methionine and phenylalanine. Another possible acceptor, pyruvate, was not examined because preliminary results showed that pyruvate was not a good acceptor in heart muscle (14). Substrate (6.6XIO- 3 M) Norvaline Norleucine TABLE V Substrate Specificity Activity (m/<mole/10min.) When the keto acids of the branched chain amino acids were incubated separately with glutamate and enzyme, there was considerable formation of ketodicarboxylic acid, possibly a-ketoglutarate, as shown in Table VI. This shows that this transaminase reaction is reversible. Crystalline soluble and mitochodrial aspartate transaminases (19) and highly purified alanine transaminase (21) from hog heart did not show any activity for branched chain amino acids.

7 166 A. ICHIHARA and E. KOYAMA TABLE VI Reversibility of the Reaction Substrate (6.6xlO" 3 M) Activity (m/imole/10 min.) Amino acid Keto acid a-ketomonocarboxylate formed a-ketodicarboxylate formed Glutamate 100 // // a-ketoglutarate // II «-Ketoisovalerate a-ketoisocaproate D-a-Keto-/3-methylvalerate VALINE, METHI0NINE OR ORNITHINE ( X I0" 3 M ) O ISOLEUCINE (xlo" 3 A/) FIG. 6. Inhibition of enzyme activity with leucine by addition of valine or isoleucine. The reaction mixture contained (in /imoles); sodium pyrophosphate buffer (ph 8.6) 50, L- leucine-1-c (16,375 c.p.m./^mole), «-ketoglutarate 10, pyridoxal phosphate 0.1, the various amounts of L-amino acids shown in the figure and enzyme in a total volume of 1.5 ml. The L-amino acids added were: A : isoleucine; : valine; A : methionine; O : ornithine l/leucine[xlo- 3 Af] Fio. 7. Competitive inhibition of enzyme activity with leucine by valine or isoleucine. The reaction mixture was the same as for Fig. 6, except that various concentrations of L-leucine-1-C 14 were added in the presence of a fixed concentration of valine (3.3xlO~ 2 /W) or isoleucine (6.6xlO" 3 M). Competitive Inhibition of Activity by Substrates When leucine-1-c 14 was incubated together with valine or isoleucine, it was found that formation of C'Mceto acid, possibly a-ketoisocaproic acid-l-c u, was inhibited progressivly with increase in the concentration of the other two amino acids (Fig. 6). Amino acids other 0.5

8 Branched Chain Amino Acids Transaminase. I 167 TABLE VII Compititive Inhibition by Substrate Amino acid added Concentration (XlO- 3 M) Total keto raonocarboxylate formed Activity (m/*mole/10 min.) a-ketoisocaproate-1 -C u formed -1-C u -1-C u 6.6 /6. 6 \ than branched chain amino acids, such as ornithine or methionine, did not show any inhibitory effect. However, the formation of total keto acid was shown to be the same whether leucine alone, or together with isoleucine, was incubated as shown in Table VII. This indicates that isoleucine was not only inhibitory for transamination of leucine, but that it was also transaminated to the extent that it was inhibitory. This competitive nature of branched chain amino acids as substrates is clearly shown in Fig. 7 in which various concentrations of leucine-1-c 14 were incubated with a constant amount of valine or isoleucine. These results also indicate that a single enzyme is responsible for the transamination of the three branched chain amino acids and that this enzyme activity is fairly specific for the amino acids. DISCUSSION It has been known for a long time that valine, leucine and isoleucine can be transaminated with fairly high activities, though less than the activities with aspartate and alanine when a-ketoglutarate was used as an acceptor (6 9). However, there are no reports on the purification and characterization of the activity for branched chain amino acids, thus in higher animals it is uncertain whether these transaminations are catalyzed by a single enzyme or by different enzymes, or whether an enzyme with broard substrate specificity is responsible for transamination of these branched chain amino acids as well as other amino acids. The present work was focused mainly on the identity of the enzyme responsible for these reactions. The results obtained in the present work indicate very strongly that valine, leucine and isoleucine are transaminated by a single enzyme, the specificity of which is limited almost completely to branched chain amino acids. This enzyme can be named branched chain amino acid : 2-oxoglutarate aminotransferase [EC 2.6.1]. This conclusion was drawn from the following findings: a. The activity ratios with the three substrates were constant during enzyme purification. b. The enzyme activities towards the three amino acids decreased in parallel during heat treatment. c. The three amino acids competed with each other as substrates when added together. Other amino acids did not have this inhibitory effect. d. The ph curves for the activity with the three substrates were similar and all showed a ph optimum at 8.6. e. The substrate specificity of the enzyme was limited to branched chain amino acids, although there was some activity with norleucine and norvaline. With regard to Neurospora there are several reports showing that branched chain amino acids are transaminated by a rather nonspecific transaminase which is also active with other amino acids, such as phenylalanine and methionine (11 13). Rudman and M e i s t e r also found that in E. coli these three amino acids are transaminated by a nonspecific enzyme (10). In animals the most abundant transaminases are aspartate trans-

9 168 A. ICHIHARA and E. KOYAMA aminase and alanine transaminase. However, neither of the two transaminases from hog heart, in the case of aspartate transaminase both soluble and mitochondrial enzymes, had any activity with branched chain amino acids. The inactivity of alanine transaminase with leucine has also been reported (21). It is interesting that these branched chain amino acids inhibit ornithine-keto acid transaminase [EC ], but they are not substrates for it (22, 23). M c i s t c r also reported asparagineketo acid transaminase [EC ] or glutamine-keto acid transaminases [EC ] in animals (24), but of the branched chain amino acids only the keto analogue of leucine is transaminated by these enzymes, and the reaction is essentially irreversible. From these considerations the transaminase reported in the present work seems very likely to be a new enzyme. Beside the soluble fraction, the mitochondrial fraction contained considerable activity for branched chain amino acids but this activity can not be extracted without addition of detergent. It was also found in preliminary work that branched chain amino acids can be transaminated with pyruvate and the activity is localized predominantly in the soluble fraction of liver among the tissues tested and that leucine was the best of the three amino acids as substrate (14). The purification and characterization of these enzymes are now under way. SUMMARY The distribution of valine, leucine, isoleucine-a-ketoglutarate transaminase activity was determined in various tissues of rats. It was found that heart and kidney were the most active organs for the activity,, followed by skeletal muscle and that liver showed very low activity. The best substrate among these three amino acids was either leucine or isoleucine depending upon the tissue used. was the poorest of the three in all tissues examined. The subcellular distribution of enzyme activity in rat heart showed that both the supernatant and mitochondrial fractions contained activity. The partial purification of soluble transaminase from hog heart was carried out and during the purification procedures the activity ratios for the three amino acids remained constant. The enzyme had an optimal ph at 8.6 for the three amino acids and the simultaneous presence of one of the three amino acids, a-ketoglutarate and pyridoxal phosphate was essential for the enzyme activity. The activity was shown to be reversible. The transaminase was inactivated by heat treatment and the activity for the three amino acids decreased at the same rate. The substrate specificity for the transaminase was chiefly limited to branched chain amino acids, but norvaline and norleucine showed lesser activities. Other amino acids examined were all inactive., leucine and isoleucine were shown to be competitive type substrates and it was concluded from these findings that the transaminase studied was specific for branched chain amino acids and the three amino acids were transaminated by the same enzyme. The authors are greatly indebted to Drs. Y. Takeda and H. Wada, of this University, for their continuous interest and valuable suggestions throughout this work. REFERENCES (/) Harper, A.E., Benton, D.A., Winje, M.E., and Elvehjem, C.A., Arch. Biochem. Biophys., 51, 523 (1954) (2) Benton, D.A., Harper, A.E., Spivey, H.E., and Elvehjem, C.A., Arch. Biochem. Biophys., 60, 147 (1956) (3) Rose, W.C., Johnson, J.E., and Haines, W.J., J. Biol. Chem., 145, 679 (1942) (4) Ringer, A.I., Frankel, E.M., and Jonas, L., J. Biol. Chem., 14, 525 (1913) (5) Butts, J.S., Blunden, H., and Dunn, M.S., J. Biol. Chem., 120, 289 (1937) (6") Cammarata, P.S., and Cohen, P.P., J. Biol. Chem., 187, 439 (1950) (7) Awapara, J., and Seale, B., /. Biol. Chem., 194, 497 (1952) (8) Rowsell, E.V., Biochem. J., 64, 235 (1956) (9) Rowsell, E.V., Biochem. J., 64, 246 (1956) (10) Rudman, D., and Meister, A., J. Biol. Chem., 200, 591 (1953) (//) Fincham, J.R.S., and Boulter, A.B., Biochem. J., 62, 72 (1956)

10 Branched Chain Amino Acids Transaminase. I 169 (12) Seecof, R.L., and Wagner, R.P., /. Biol. Chem., 234, 2689 (1959) (13) Seecof, R.L., and Wagner, R.P., /. Biol. Chem., 234, 2694 (1959) (14) Ichihara, A., and Koyama, E., ' Proceedings of the Symposia on Enzyme Chemistry ' (in Japanese), 17, 337 (1965) (15) Wada, H., and Snell, E.E., j. Biol. Chem., 237, 127 (1962) (IS) Meister, A., "Biochemical Preparations ", John Wiley and Sons Inc., New York, Vol. m, p. 66 (1953) (17) Hogeboom, G.H., " Methods in Enzymology ", ed. by S.P. Colowick and N.O. Kaplan, Acad. Press Inc., New York, Vol. I, p. 16 (1955) (18) Takeda, Y., Ichihara, A., Tanioka, H., and Inoue, H., J. Biol. Chem., 239, 3590 (1964) (19) Wada, H., and Morino, Y., Vitamins and Hormones, 22, 411 (1964) (20) Katunuma, N., Mikumo, K., Matsuda, M., and Okada, M., J. Vitaminol., 8, 68 (1962) (21) Segal, H.L., Beattie, D.S., and Hopper, S., J. Biol. Chem., 237, 1914 (1962) (22) Katunuma, N., Matsuda, Y., and Tomino, I., J. Biochem., 56, 499 (1964) (23) Strecker, H.J., J. Biol. Chem., 240, 1225 (1965) (24) Meister, A., Sober, H.A., Tice, S.V., and Fraser, P.E., J. Biol. Chem., 197, 319 (1952)