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Amino Acid Metabolism in Microorganisms Part IV L

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Amino Acid Metabolism in Microorganisms Part IV L [Agr. Biol. Chem., Vol. 32, No. 6, p. 727-733, 1968] Amino Acid Metabolism in Microorganisms Part IV L-Methionine Decarboxylase Produced by a Streptomyces Strain By Hiroshi HAGINOand Kiyoshi NAKAYAMA Tokyo Research Laboratory, Kyowa Hakko Kogyo Co., Ltd. Received December 1, 1967 3-Methylthiopropylamine (MTPA) formation from L-methionine in Streptomyces sp. K37 was studied in detail. The reaction was confirmed to be catalyzed by the decarboxylase of L-methionine. The properties of the enzyme were studied in detail using acetone dried cells or cell-free extract. The enzyme was specific for L-methionine. Pyridoxal phosphate stimulated the reaction and protected the enzyme against heat inactivation. The optimum pH for the reaction was 6.0-8.0 and the optimum temperature was about 40°C. Carbonyl reagents (10-2-..10-3 M) inhibited the reaction completely, and silver nitrate and mercuric chloride (10-3•`10-4M) markedly inhibited the reaction. Km value for the reaction was 1.21 X 10-5 M. L-Methionine assay using the decarboxylase was attempted and was found to be applicable to practical use. As previously reported", certain Streptomyces using acetone dried cells or cell free extract strains isolated from soil accumulated 3- of Streptomyces sp. K37. L-Methionine assay methylthiopropylamine (MTPA), decarboxy using this decarboxylase was also attempted. lated product of methionine, when they were This paper deals with the results of these cultured in the medium containing L- or DL- experiments. methionine. Now, MTPA formation from L-methionine by Streptomyces sp. K37 was MATERIALS AND METHODS further studied in detail. MTPA formation Strains. An unidentified strain, Streptomyces sp. was confirmed to be the reaction catalyzed by K37 isolated from soil was used. L-methionine decarboxylase (L-methionine Culture method. Thirty millilitre of a seed carboxylyase). As far as authors know, medium in a 250 ml Erlenmyer flask was inoculated enzymic decarboxylation of L-methionine has with the spores from a Bennett-agar slant and never been reported though oxidative decar incubated at 30°C for 48 hr. The seed medium boxylation of L-methionine was studied in contained 2% glucose, 1%, yeast extract, 1% cabbage"" and horse raddish4,5) by M. peptone, and 0.25 0 sodium chloride (pH 7.4 before sterilization). The seed culture was transferred into Mazelis. Therefore, the properties of the a 250 ml Erlenmeyer flask containing 20 ml of a decarboxylase of L-methionine was examined growth medium and the flask was incubated at 30°C on a rotary shaker for 96 hr. The growth medium 1) H. Hagino and K. Nakayama, Agr. Biol. Chem., had the following composition: glucose 1%, K2HPO4 31, 1369 (1967). 0.05 00, MgSO4•E7H2O 0.025%, (NH4)2SO4 0.2% 2) M. Mazelis, Biochem. Biophys. Res. Commum., 1, 59 (1959). yeast extract 0.3 0„ CaCO3 0.3 0,,' and L-methionine 3) M. Mazelis, Federation Proc., 19, 4 (1960). 5 mg/ml (pH 7.4 before sterilization). 4) M. Mazelis, J. Biol. Chem., 237, 104 (1962). Preparation of acetone dried cells or cell free 5) M. Mazelis and L. L. Ingraham, ibid., 237, 109 (1962). extract. The cells grown in the growth medium 728 Hiroshi HAGINO and Kiyoshi NAKAYAMA for 6-8 hr were harvested and washed twice with TABLE I. STOICHIOMETRY OF DECARBOXYLATION saline water, and then acetone dried cells were OF L-METHIONINE prepared by treating the washed cells with cold acetone. The washed cells were suspended in 1/15 M phosphate buffer (pH 6.0) to be the cell concentration 10---15 mg (dry weight) per millilitre. The cells were disrupted by sonic treatment for 20 min at 0°C with a Ultra Sonic Oscillator (Kaijo Denki Co.). Cellular debris was removed by centrifugation at 15,000 X g for 30 min, and the clear supernatant fraction was obtained. Then, ammonium sulfate Reaction mixture: acetone dried cells (60mg/ml) 1 ml, 1/15M phosphate buffer (pH 6.0) 1.3ml, was added to the supernatant in a concentration of L-methionine (3-5 mg/ml) 0.5 ml, (4.5 N HCl 0.2 50% saturated. The preciptate obtained was ml) dissolved into 0.01 M phosphate buffer and dialyzed Reaction was carried out at 37.5°C for 10 min. against the same buffer for 18 hr at 5•Ž. The dia lyzed fraction was used for the enzyme assay. from L-methionine accompanied equimolecular Enzyme assay. Enzyme activity was determined CO, liberation. When the decarboxylation manometrically by measuring CO2 liberation by a reaction was carried out in the reaction Warburg manometer or spectrophotometrically by deter- mining MTPA produced. In the manometeric method, mixture containing 0.2 ml of 20 % KOH in the main compartment contained the enzyme solution the centre well, CO, liberated was absorbed (0.5---1.0 ml)with appropriate amount of buffer (1.3- into KOH and little gas evolution was 1.5 ml) and pyridoxal phosphate solution (0.3ml), a observed. These facts suggest that the side-arm contained L-methionine solution (0.5 ml) and reaction is simple decarboxylation of L- the other side-arm contained 4.5 N HC1 (0.2 ml). The methionine other than the oxidative decar total volume of the reaction mixture was 3.0 ml, boxylation reported by M. Mazelis2•`5) The reaction was cerried out at 37.5"C. To liberate 2. Decarboxylase activity and residual L-me the retained CO2, 4.5 N HCL (0.2 ml) was tipped into thionine. the reaction mixture at the end of the experiment. As previously reported", MTPA forming In the assay of L-methionine by the decarboxylase method, acid tip was not used and the CO2 retained activity was induced in the existence of L- was corrected by calculation. In the photometric methionine in the growth medium and was method, MTPA formed was measured by ninhydrin found in the cells incubated for short period reaction according to the modified method of Giris) (6-8 hr) but little activity was found in the after isolating the MTPA from residual L-methionine cells incubated for longer period. Therefore, by paperchromatography using the solvent system; the decarboxylase activity in the cell was n-butanol: acetic acid: water (4: 1:2). A minute examined in relation to the concentration of amount of MTPA was determined by the color residual L-methionine in the broth. As shown development with picric acid according to the in Fig. 1, the decarboxylase activity in the Richter's method7) after extracting the MTPA from cell decreased in parallel with the rapid con the reaction mixture at alkaline pH with ether. sumption of L-methionine in the medium. RESULT New supplement of L-methionine to the broth 1. Stoichiometry of MTPA formation from L- after 6, 12, and 24 hours' incubation retained methionine. or increased the decarboxylase activity of the As shown in Table I, MTPA formation cell. 3. Substrate specificity. 6) K. V. Girl, A. N. Radharishnan and C. S. V L-Methionine decarboxylase in Streptomyces ardyanathan, Anal. Chem., 24, 1677 (1952). 7) D. Richter, M. H. Lee and D. Hill, Biochem. J sp. K37 was tested for the substrate specificity ., 35, 1225 (1941). with acetone dried cells. As shown in Table Amino Acid Metabolism in Microorganisms. Part IV 729 Therefore, the enzyme was confirmed to be the decarboxylase specific to L-methionine . 4. Optimum pH Fig. 2 shows the velocities of decarboxyla tion with acetone dried cells in different reaction pH. The optimum pH was 6--8 . FIG. 1. Variation of the Enzyme Activity with Age of Culture. : Enzyme activity of the cells supplemented L-methionine (5 mg/ml) to the culture broth (initial L-methionine conc. was 5mg/ml) after 6, 12, and 24 hours' incubation. FIG. 2. Effect of pH on the Activity of L-Methio -•›-: Enzyme activity of the cells without nine Decarboxylase. new supplement of L-methionine. TABLE II. SPECIFICITY OF THE L-METHIONINE DECARBOXYLASE Reaction mixture: acetone dried cell (45 mg/ml) 1ml, buffer solution 1.3ml, L-methionine (6 mg/ml) 0.5ml, (4.5N HCl 0.2ml). Reaction was carried out for 10 min at 37.5°C. Reaction mixture: acetone dried cells (60mg/ml) 1.0ml, 1/15M phosphate buffer (pH 6.0) 1.3ml, amino acid solution (5mg/ml) 0.5ml, (4.5 N-HCl 0.2ml) FIG. 3. Effect of Temperature on the Activity of Reaction was carried out at 37.5°C for 10 min. L-Methionine Decarboxylase. Reaction mixture: enzyme solution 0.5 nil, 1/15M II, only L-methionine among 22 amino acids phosphate buffer (pH 6.0) 1.7ml, L-methionine tested was decarboxylated and the small (6mg/ml) 0.5 ml, pyridoxal phosphate (50ƒÊg/ml) 0.3ml. quantity of gas evoluted from some other amino Reaction was carried out at different temperature acids may be due to experimental error. 10 min. ---•›--- 730 Hiroshi HAGINO and Kiyoshi NAKAYAMA Subsequent experiments were carried out at TABLE IV. EFFECT OF ENZYME INHIBITORS (I) pH 6.0 (1/15M phosphate buffer). 5. Optimum temperature. Fig. 3 shows the variation of the velocity of decarboxylation with temperature. The optimum temperature for the reaction was approximately 40°C. 6. Stimulation of the decarboxylation by pyridoxal phosphate. Participation of pyridoxal phosphate as a cofactor was reported in the known amino acid decarboxylation. Therefore, the effect of pyridoxal phosphate on the decarboxylation Reaction mixture: enzyme solution 0.5ml, 1/15M of L-methionine was tested using dialyzed phosphate buffer (pH 6.0) 1.65 ml, L-methionine enzyme preparation. As shown in Table III, (5mg/ml) 0.3ml, inhibitor solution 0.2ml, pyridoxal phosphate (50 fig/ml) 0.15ml (4.5 N HCl pyridoxal phosphate markedly stimulated the 0.2ml) decarboxylation.
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