Highly purified methioninase from Pseudomonas putida, which catalyzes a, 7"-elimina- tion reactions of homocysteine and its S-substituted derivatives as well ...
/. Biochem., 78, 1105-1107 (1975)
PRELIMINARY COMMUNICATION
Exchange Reactions Catalyzed by Methioninase from Pseudomonas putida Susumu ITO, Taro NAKAMURA, and Yoshitomo EGUCHI Department of Microbial Engineering & Technology, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060
Highly purified methioninase from Pseudomonas putida, which catalyzes a, 7"-elimination reactions of homocysteine and its S-substituted derivatives as well as a, ^-elimination reactions of cysteine and its derivatives, was found to catalyze exchange reactions between the substituent at the ^-carbon of homocysteine substrates and exogenously added alkanethiols, forming the corresponding S-alkylhomocysteines. It also catalyzed similar ^S-exchange reactions between cysteine and alkanethiols. Thus, all the substrates for the methioninase-catalyzed elimination reactions also appear to be available for the exchange reactions.
As described in a preliminary communication (1) and to be reported in detail elsewhere, we have purified methioninase from Pseudomonas putida ATCC 17453 (PpGl). The purified enzyme appeared homogeneous on gel-electrophoresis. We also showed that this enzyme catalyzes a, ^-elimination reactions of homocysteine and its S-substituted derivatives, such as methionine and ethionine, according to the equation: RSCH2CH2CHNH2COOH + H2O > RSH + CH3CH2COCOOH + NH 3 , where R is H, or an alkyl, or substituted alkyl group. The enzyme can also catalyze a, /3-elimination reactions of cysteine and its S-substituted derivatives as follows: RSCH2CHNH2COOH + H2O > RSH+CHjCOCOOH+NHg. Abbreviations: MET, 2-mercaptoethanol; HEH, Shydroxyethylhomocysteine. Vol. 78, No. 5, 1975
This paper reports that the enzyme also catalyzes exchange reactions between the RSgroup of homocysteine and cysteine derivatives with several alkanethiols. The occurrence of such exchange reactions was first suggested by the observation that addition of 1 mM 2-mercaptoethanol (MET) to the methioninase assay system caused about 25% decrease in a-ketobutyrate formation from methionine, and increase in production of CH3SH to 140% of the control level. When this reaction mixture was analyzed by ascending paper chromatography using 1-butanolacetic acid-water (40 : 6 : 15, by volume) as solvent, another ninhydrin-positive spot was detected besides that of residual methionine (Fig. 1, bottom). This spot also gave a positive reaction with chloroplatinic acid (2) but a negative reaction for sulfhydryl groups with nitroprusside and 5,5'-dithio-bis (2-nitrobenzoic acid), indicating that it was an amino
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Received for publication, August 16, 1975
1106
PRELIMINARY COMMUNICATION
instance, incubation of methionine and CH3CH2SH with the enzyme yielded a product which was identified as ethionine by paper chromatography in three different solvent systems. From these findings, it is concluded that the unidentified product formed from methionine and MET must be S-hydroxyethylhomocysteine (HEH). Paper chromatography showed that the formation of HEH from radioactive methionine required the presence of MET, pyridoxal phosphate, and the active enzyme. The maximum exchange activity was observed at pH 8.5, which is also the optimum pH for the elimination reaction, although a broader pH-activity curve was obtained for the exchange reaction. In an experiment on the stoichiometry of the reaction using radioactive TABLE I. Exchange reactions by methioninase. The reaction mixture contained, in 1.0 ml, 5 mM amino acid substrate (as L-isomer), 50 pM pyridoxal phosphate, 0.1 M potassium pyrophosphate buffer (pH 8.5), 1 mM alkanethiol, and 5 fig of the enzyme. After incubation for 30 min at 30°, the reactions were terminated by placing the mixtures in boilling water for 2 min. Samples of 10-50 [i\ were applied to filter paper for chromatography. Newly formed amino acids were located spraying the paper with ninhydrin and chloroplastinic acid. Ingredients of the reaction
DISTANCE FROM ORIGIN (cm)
Met.S-
Met.
Fig. 1. Formation of sulfur-containing amino acid with new substituent. The experimental conditions were as for Table I except that the added thiol was MET. The formation of a sulfur-containing amino acid from L-[U-"C]methionine and unlabelled MET was ascertained by ascending paper chromatography (Toyo No. 50) using 1-butanol-acetic acid-water (40: 6 : 15, by volume) as solvent system. Then the paper was sprayed with ninhydrin and cut into strips 1-cm width for measurement of its radioactivities.
Rf
values of newly formed amino acids a
Solvent 1
Solvent 2
Solvent 3
Methionine + CH 3CH 2SH
0.76
0.62
0.71
Ethionine+ CH3SH
0.68
0.49
0.62
Homocysteine + CH3SH
0.68
0.49
0.63
Homocysteine+ CH3CH2SH
0.76
0.61
0.71
Authentic methionine
0.69
0.48
0.63
Authentic ethionine
0.76
0.61
0.71
a
Solvent 1, 1-butanol : acetic acid : water = 5 : 2 : 3 ; Solvent 2, 1-butanol : acetic acid : water=40 : 6 : 15; Solvent 3, 1-butanol : 2-propanol : pyridine : water= 4 : 5 : 3 : 6 (by volume).
/ . Biochem.
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acid with a non-sulfhydryl sulfur. When the same experiment was repeated using h-[U-uC] methionine and unlabelled MET as substrates, most of the radioactivity was recovered in the areas corresponding to the unidentified product and residual methionine (Fig. 1, top); the two small radioactive peaks at i?/=0.14 and i?/=0.69 were identified as methionine sulfoxide (formed from methionine during chromatography) and a-ketobutyrate, respectively. These results suggested that the unidentified product was formed by an exchange of the —SCH3 group of methionine with MET. Thus it seemed interesting to examine whether the enzyme could catalyze similar exchange reactions between methionine, ethionine, and homocysteine on the one hand and CH3SH and CH3CH2SH on the other. Table I shows that these exchange reactions were detected with all four combinations of sulfur amino acids and thiols tested. For
EXCHANGE REACTIONS BY METHIONINASE
methionine and unlabelled MET as substrates, 0.58 /rnole of CH3SH was formed with accumulation of 0.23 ^mole of a-ketobutyrate and 0.33 pmole of HEH. Thus under the conditions employed about 40% of the methionine was consumed in the elimination reaction and about 60% in the exchange reaction. From the above results and our preliminary finding that methioninase catalyzed the formation of S-methylcysteine from cysteine and CH3SH, we conclude that this enzyme •can catalyze the a, y- and a, /3-elimination reactions mentioned above and also following •y- and /3-exchange reactions:
and RSCH2CHNH2COOH + R'SH ^ R'SCH2CHNH2COOH+RSH (/8-exchange), where R'SH is an exogenously added alkanethiol. In this connection it is noteworthy that bacterial enzymes known to catalyze /3-elimination type reactions, such as cysteine de•sulfhydrase [EC 4.4.1.1] (3), tryptophanase [EC 4.1.99.1] (4-6), and ^-tyrosinase [EC 4.1.99.2] (7, 8) have also shown to catalyze .a series of /8-exchange reactions (5, 6, 9—11). In the course of preparation of our manuscript, similar results were reported by Ezaki, Tanaka, Yamamoto and Souda.1 1 Presented at the Annual Meeting of the Agricultural Chemical Society of Japan, July 24, 1975, Sapporo.
Vol. 78, No. 5, 1975
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RSCH2CH2CHNH2COOH+R'SH ;=± R'SCH2CH2CHNH2COOH+RSH (j-exchange)
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