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The Journal of Biochemistry, Vol. 33, No. 4, ISO

Metabolism of Methyl Saccinate in Pseudomonas fluorescena Grown on Itaconate

Methyl succinate has been reported to be oxidized by crude succinate dehydrogenase preparation from horse heart (1% guinea pig liver mitochondria (2) and the resting cells of Ps.fluorescensgrown in the presence of itaconate (5, 4). Although mesaconate was detected in the urine of dogs fed with methyl succinate (5), none of the degradation products from methyl succinate have been reported in cell-free systems. The resting cells of Ps-fluoresctm No. 3081, aerobically grown on a culture medium containing 0.3 per cent of itaconate and 0.2 per cent of glucose as carbon sources,rapidly oxidized DL-methyl succinate, citramalate, mesaconate besides itaconate. The amounts of Oi consumed and COj liberated during the oxidation of DL-methyl succinate were about 56 per cent of the calculated values for the complete degradation, and they were equal to approximately the values of itaconate, mesaconate and citramalate (only D-isomer is supposed to be metabolized in the case of citramalate).* The cell-free extract was prepared by disrupting the cells in a sonic disintegrator for three minutes in Af/15 phosphate buffer (pH 73), followed by a centrifugation at 12,000 xg for 40 minutes. The cell-free preparation was incubated with 4-Cu-methyl succinate in the presence of ATP, MgClt and phenazine methosulfate in a Warburg vessel. After the incubation period, the reaction was stopped by the addition of sulfuric acid from the

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Formation tf Jtadioaetwt Compounds from t-O'-Mtthjrl SueeinaU




MS" remained (c.p.m.) 222 6,740 7,130 4,380

Radioactivity of product (c.p. m.)





1. 2.

(1) (2) (3)

Crude extinct of IA-Cell" Crude extract of Gl.-Cell" Cleavage enzyme"-t-boiled (1) Cleavage enzyme+(1)


< 10 < 10 1,575

503 < 10 < 10 40

28 50 < 10 25

1,090 35 < 10 340

The reaction mixture contained (janoles), phosphate buffer (pH 7.3) 200; ATP, 10; MgCl,, 15; phenazine methosulfate, 5; 4-DL-Cu-methyl tuccinate, 8 (820 c.p.m./^mole, synthesized from KC14N and ethyl methacrylate (7)) and enryme preparation. Incubations were carried out in a total volume of 2.6 ml., at 30*C for 30 (Expt. 1) and 60 (Expt. 2) minutei. After the reaction mixture wai deproteinized, CoA derivatives were hydrolyzed (5). The radioactivities were measured with a thin window gas-flow counter. 1) Abbreviations: MS, methyl succinate; A A, acetate; LA, lactate; CM, citramalate. 2) During the procedure preparing the sample for column chromatography, a significant loss of acetate was observed. In an experiment with authentic radioactive acetate, the recovery of radioactivity through the procedure was about 80 per cent. As the conditions were not exactly the same, no corrections for the insufficient recovery were made. 3) Crude extract of itaconate-grown cells. 38mg. of protein. 4) Crude extract of glucose-grown cells. 45mg. of protein. 5) Citramalyl-CoA lyase (9) purified about 20-fold from itaconate-grown cells. 4.6 mg. of protein. Kauuki, F., in preparation. 328

Methyl Sucdnatc Metabolism side arm and C H O t formed was absorbed into alkali in the center well. The reaction mixture was deproteinized and dried up in vacua. The samples were then applied to a celite column and the incorporation of radioactivity into organic acids were investigated according to the method of P h a r e s tt al. (6). As is shown in Table I, almost all of DL-4C^-methyl succinate disappeared and the radioactivity was mainly incorporated into acetate and carbon dioxide. Small amount of incorporation was also found in the fractions of lactate (or succinate) and of citramalate. Radioactive acetate was identified by rechromatography with authentic acetate, paper chromatography and recrystalization as /^-bromophenacyl ester. Citramalate was identified by paper chromatography. The product isolated in the fractions of lactate and succinate were tentatively identified to be lactate, as a small amount of incorporation into pyruvate was also demonstrated when the incubation was carried out in the presence of semicarbazide. Without the addition of phenazine methosulfate, the degradation of methyl succinate was also demonstrated "but its rate was slower. The amount of incorporation of radioactivity into amino acids (by Dowex-50, H+-form column) was about one tenth of that incorporated into acetate. No radioactive product was detected either by crude extract of glucose-grown cells or by the partially purified citramalyl CoA synthase* from itaconate-grown cells (0). But when the crude extract was mixed with the cleavage enzyme and incubated with methyl succinate, jadioactive acetate was produced. (Expt. 2).
COOH CH-CH, CH, , COOH . C-CH, CH <A> . ! . - _ . COOH TTl HOC-CH, CH, (B) COOH citramalic add (C)


This fact suggests a participation of cleavage enzyme in the methyl succinate metabolism. From these data, the main metabolic path of methyl succinate in the microorganism is supposed as shown in the following scheme. The presence of mesaconate hydratase** (B) (10), citramalyl-CoA synthetase*** (C) and citramalyl-CoA lyase (D) (9) have been demonstrated in this microorganism. Among them, only the cleavage enzyme is induced by itaconate (9). The formation of radioactive mesaconate expected from the scheme could not be demonstrated, probably because of the low specific radioactivity of the substrate used and of the inhibiting action of mesaconate added to trap the radioactivity. The degradation of methyl succinate was also inhibited by itaconate or citramalate. These facts might also be useful to explain why radioactive intermediates could not be demonstrated in the glucose-grown cells. The formation of C14Oj seems to be caused by the further oxidation of acetyl-CoA. Up to now, the process of the formation of Cu-lactate has been unknown and the investigation to demonstrate the reversibility of the reaction (A) (11) has been unsuccessful.

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Franke, W., and Siewerdt, D., Z. physiol. Chtm. 280, 76 (1944) (2) Adler, J. Wang. S-F., and Lardy, H. A . , / . Biol. Clum., 229, 865 (1957) ( 3 ) Kauuki, H., Nagai, J., Katsuki, F.( Egashira, S., and Tanaka, S., Symposia on Entymt C/umistry 16, 128 (1962> (*) Brightman, V., and Martin, W. R . , / . Bacteriol., COOH COOH ; = CO CH, pyruvic acid acetyl-CoA CH,


7> HOC-CH,


I (D) CO.SCoA dtramalyl-CoA


C f


methyl succinic acid

mexaconic acid

* The enzyme may be grouped in ketoacid lyase XEC 4. 1. 3]

* EC 4. 2. 1 Group (hydro-lyaie) ** EC 6. 2. 1 Group (acid-thiol ligaje)

(5) (6)

Letters to the Editors

Biol. Chtm., 236, 26 (1961) (9) Nagai, J., J. Biochrm., in the press (10) Katsuki, H., Ariga, N., Katsuki, F., Nagai, J., Egajhira, S., and Tanaka, S., Biochim. it Bicphys. Atta, 56, 545 (1962) (11) Fisher, F. G., and Eysenbach, H., Am. Chtm., 530, 99 (1937)

82, 376 (1961) Emmrich, R., Z. physiol. Cfum., 261, 61 (1939) Phares, E. F., Mosbach, E. H., Denison, F. W. Jr., and Canon, S. F., Anal. Chtm., 24, 660 (1952) ( 7 ) Brown, B., Org. Synthtsis, 26, 54 (1946) (8) Wang, S-F., Adler, J., and Lardy, H. A., / .

Department of Chemistry, Faculty of Science, Kyoto University, Kyoto


(Received for publication, January 16, 1963)

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