Agric. Biol. Chem., 41 (1), 169174, 1977

Purification and Properties of Methylisocitrate , a Key in Propionate Metabolism, from Candida lipolytica

Takeshi TABUCHI and Takayori SATOH*

Institute of Applied Biochemistry, The University of Tsukuba, Ibaraki-ken 300-31, Japan *Faculty of Agriculture , Tokyo University of Education, 2-19-1, Komaba, Meguro-ku, Tokyo Received August 24, 1976

Methylisocitrate lyase catalyzing the cleavage of threo-Ds-2-methylisocitrate into pyruvate and succinate was purified about 60-fold from cell free extracts of Candida lipolytica. The purified enzyme required a divalent cation and a sulfhydryl compound for maximum activity. Sulfhydryl reagents strongly inhibited enzyme activity. The molecular weight was estimated to be about 1.2 x 101 by gel filtration. The enzyme was specific for threo-Ds-2-methyliso citrate (Km=7.7 x 10-4 M) and did not catalyze the cleavage of threo-DL-isocitrate. Attempts to detect condensation between pyruvate and succinate by the preparation were unsuccessful. The enzyme was activated by NAD but noncompetitively inhibited by NADH and NADPH: these results support the idea that this enzyme is also a regulatory enzyme of the methylcitric acid cycle concerning the oxidation of propionate to pyruvate.

We have proposed the methylcitric acid from C. lipolytica. The control mechanism of cycle concerning the partial oxidation of pro- the cycle is also discussed from the results of pionyl-CoA into pyruvate via seven-carbon inhibition studies. tricarboxylic acids (Fig. 1) in yeasts1'2) and presented evidence in support of this cycle.3•`7) MATERIALS AND METHODS Methylcitrate synthase, functioning at the and medium. C. lipolytica IFO 1659 entrance of this cycle (I in Fig. 1), is shown to was used for preparing enzyme. A medium for cul differ from the usual citrate synthase (EC tivation consisted of glucose 8 %, sodium propionate 4.1.3.7, citrate oxaloacetate-lyase (CoA-acetyl- 1 %, urea 0.3 % (sterilized separately), KH2PO4 0.05 %, ating)) of Candida lipolytica.8) In the preceding MgSO4' 7H2O 0.05 %, and yeast extract 0.1 % in tap study, the other key enzyme (III in Fig. 1), water. cleaving threo-Ds-2-methylisocitric acid Culture. Cells grown on an potato-glucose agar (MICA), has been separated from the usual slant were inoculated into ten of 500-m1 Erlenmeyer (EC 4.1.3.1 threo-Ds-isocitrate flasks containing 50 ml of the medium and incubated for 2 days at 26°C on a rotary shaker at 220 rpm. glyoxylate-lyase) of C. lipolytica by DEAE All the culture broths were then transferred into a cellulose chromatography, and given the trivial 10-liter jar fermentor containing 5 liters of the medium. name of methylisocitrate lyase; the two en Cells were grown under the conditions of the agitation zymes differ in their specificities, of 800 rpm and the aeration of 1 liter per min at 26°C chromatographic elution patterns, pH optima, for 2 days. Cells were harvested by centrifugation, kinetics of thermal inactivations, and behavior washed twice with 0.01 M Tris-HCl buffer, pH 7.5, containing 3 mm MgC12 and 1 mm 2-mercaptoethanol with inhibitors.9) (hereafter referred to as TMM buffer), and stored at This paper describes the purification and -20°C. some properties of the methylisocitrate lyase Enzyme assay. Methylisocitrate lyase was assayed Abbreviation: MICA, threo-Ds-2-methylisocitric by the continuous spectrophotometric method as acid; TMM buffer, 0.01 M Tris-HCl buffer, pH 7.5, described in the preceding paper9); one unit of activity containing 3 mm MgC12 and 1 mm 2-mercaptoethanol. was expressed in nanomoles of pyruvate phenylhydra- 170 T. TABUCHI and T. SATOH

additional ammonium sulfate (4 g/ml) was added to the supernatant to achieve 56%. saturation. After the mixture had been allowed to stand for 45 min, the precipitate was collected by centrifugation as before. Step 3. Sephadex G-150 chromatography. The precipitate was dissolved in 6 ml of TMM buffer. This solution was placed on a Sephadex G-150 column (100 by 2.4 cm) equilibrated beforehand with TMM buffer, and the enzyme was eluted (overnight) with TMM buffer. Fractions (10 g) were collected and assayed for enzyme activity. Step 4. Second ammonium sulfate fractionation. To the pooled fractions (30 ml) with high enzyme ac tivity were added ammonium sulfate to give 56% saturation. After the mixture had been allowed to stand for 45 min, the precipitate was collected by centrifugation at 5,000 x g for 20 min, dissolved in 20 ml of TMM buffer, and the solution was dialyzed overnight against TMM buffer. Step 5. DEAF-cellulose chromatography. The dia lyzed solution was charged onto a DEAE-cellulose column (30 by 2.0 cm) equilibrated with TMM buffer. This column was washed with 200 ml of TMM buffer, and the protein was eluted by a linear Tris-HCI gradient increasing from 0.01 M to 0.2 M in 200 ml of TMM zone formed from MICA per min at 25°C. Specific buffer. The column was run overnight and 3 g activity was expressed as units per mg of protein. fractions of eluates were collected and assayed. Frac Protein was determined by the method of Lowry et al.10) tions having enzyme activity were combined, and con centrated in a collodion bag apparatus to a final volume Purification of methylisocitrate lyase of 10 ml. The concentrated enzyme was then divided Step 1. Autolysis. Toluene (16 ml) was added to into small batches and stored at -20°C. the cells (260 g as a pasty cake), and the mixture was

incubated with occasional stirring for 45 min at 37•Ž. Molecular weight estimation. Gel filtration was The autolyzate was treated just as described previously.8) performed with columns of Sephadex G-150 and G-200

After centrifugation at 11,000 x g for 20 min, the super- columns (100 by 2.4 cm) equilibrated with TMM buffer, natant of the autolyzate was brought to pH 7.5 with and 5 g fractions were collected. 5 N KOH. All subsequent steps were carried out at Disc gel electrophoresis. Electrophoresis of the 0 to 5°C. purified enzyme preparation was performed with a Step 2. First ammonium sulfate fractionation. The 7.5 % polyacrylamide gel and TMM buffer by the supernatant from the preceding step, referred to as method of Davis11) at a current of 2 mA/gel for 2 hr autolyzate in Table I, was adjusted to 42% saturation at 4°C. with solid ammonium sulfate (3 g/ml), and the mixture was allowed to stand for 45 min. After removal of the Reverse reaction. The composition of reaction precipitate by centrifugation at 5,000 x g for 20 min, mixture used in order to test for the occurrence of condensation between pyruvate and succinate was as TABLE I. SUMMARY OF PURIFICATION follows: potassium pyruvate, 10 µmoles; sodium suc cinate, 10 µmoles; MgCl2, 10 µmoles; cysteine-HCI, 10 µmoles; phosphate buffer, at pH 6.5, 7.0 or 7.5, 200 µmoles; and the enzyme preparation, 0.5 units in a total volume of 1 ml. After incubation for 20 hr at 30°C, the reaction was examined by gas chromatography as described previously.7)

Chemicals. The protein markers and blue dextran used in gel filtration were purchased from Boehringer a) Expressed as nanomoles of product formed per Mannheim GmbH and threo-DL-isocitrate from Wako minute per mg of protein. Chemical Industries, Osaka. All other chemicals were Properties of Methylisocitrate Lyase 171 obtained as described previously .7,8) TABLE II. EFFECT OF COFACTORS ON ENZYME ACTIVITY

RESULTS Purification of methylisocitrate lyase Table I shows the results of a purification run. The enzyme preparation obtained in step 5 of Table I represents about a 60-fold purification with a recovery of about 4 % of the original activity. The enzyme preparation showed a single TABLE III. MICHAELIS CONSTANTS OF REACTANTS protein band (Rf 0.10), which coincided with the band of the enzyme activity, in polyacryl amide gel electrophoresis. The enzyme preparation contained no detect- able amounts of isocitrate dehydrogenases (NAD-specific (EC 1.1.1.41) and NADP specific (EC 1.1.1.42)),12) malate dehydro cation and a sulfhydryl compound for maxi- genase (EC 1.1.1.37),13) aconitate hydratase mum activity (Table II). Lineweaver-Burk (EC 4.2.1.3),14) or isocitrate lyase (EC 4.1. plots for MICA (0.1 to 0.5 mm), Mg2+ ion 3.1).15) (0.05 to 0.2 mm), and cysteine (3 to 20 mm) were linear. Michaelis constants are given in Table Molecular weight III. The molecular weight of methylisocitrate lyase was between 1.1 x 105 and 1.3 x 105 as Effects of temperature and pH on enzyme determined by gel filtration methods using activity Sephadex G-150 and G-200 (Fig. 2). The effects of temperature and pH on enzyme activity were examined by the standard assay method except that temperature and buffer solutions were changed. The optimum temperature for enzyme activity was found to be at about 40°C (Fig. 3A) and the optimum

FIG. 2. Molecular Weight of Methylisocitrate Lyase. -, Sephadex G-150; ------, Sephadex G-200; O, cytochrome c; G, myoglobin; O, ovalbumin; A, albumin; •, methylisocitrate lyase; o, gamma- globulin. Ve, Elution volume of protein; Vo, void volume of column determined with blue dextran. FIG. 3. Effects of Temperature (A) and pH (B) on requirement Enzyme Activity. Methylisocitrate lyase required a divalent ------, phosphate buffer; , Tris-HC1 buffer. 172 T. TABUCHI and T. SATOH pH between 7.0 and 7.7 (Fig. 3B). Sharp All attempts to detect condensation be decreases in activity were observed as the pH tween pyruvate and succinate by the enzyme deviated from the optimum pH; the effect of preparation ended in failure. pH was the same as described in the preceding paper." Stimulatory and inhibitory effects of metallic ions Effects of heat and pH on enzyme stability Since the stimulation of enzyme activity was The enzyme preparation was incubated with observed in the presence of Mgt+, other TMM buffer at various temperatures for divalent cations were tested to determine 10 min, and remaining activities were meas their effects. As Table IV shows, several ca ured. As Fig. 4A shows, enzyme activity was tions activated methylisocitrate lyase but fairly stable at 40°C but completely lost at Mg2+ was the most effective metallic ion as 60°C. the activator under the experimental conditions employed. Under the conditions of coexistence with Mg2+ (2.5 mm), Cu2+, Hg2+ and Zn2+ at a concentration of 10 mm inhibited the enzyme activity in some extent (Table IV).

TABLE IV. EFFECT OF METAL IONS ON ENZYME ACTIVITY

FIG. 4. Effectsof Temperature(A) and pH (B) on EnzymeStability. ------, phosphatebuffer; -, Tris-HClbuffer.

After incubation of the enzyme preparation with Tris-HC1 and phosphate buffers of ° Metal ions were added at a concentration of various pH values (final buffer concentration, 2.5 mm in stead of Mg2+. 0.02 M) at 15°C for 20 hr, each of the mixtures '~ Metal ions were added at a concentration of was diluted 5-fold with 0.1 M Tris-HCl buffer 10 mm in addition to Mg2+ (2.5 mm). (pH 7.5) containing 3 mm MgC12 and 1 mm 2-mercaptoethanol, and remaining activities Inhibition studies were measured. As Fig. 4B shows, enzyme Several compounds were tested as possible activity was fairly stable at pH 7.5. inhibitors (Table V). All sulfhydryl reagents tested showed substantial inhibitory effects on Substrate specificity the enzyme activity. On the contrary, any of The enzyme preparation did not seem to organic acids tested had little or no effect at a cleave threo-Ds-isocitrate, threo-DL-isocitrate, concentration of 1 mm, except threo-DL-iso and erythro-Ls-isocitrate: considering the sensi- citrate; it was tested at twice the concentration tivity of the assay method employed, we esti to check the effect of threo-Ls-isocitrate. Keto mated the rate of these isocitrates cleavage by acids were not tested because the present the enzyme, if present, only less than 0.5 % of standard assay mixture contained phenyl that of threo-Ds-2-methylisocitrate cleavage. hydrazine. Properties of Methylisocitrate Lyase 173

TABLE V. EFFECT OF VARIOUS COMPOUNDS ON ENZYME ACTIVITY

FIG. 6. Stimulation or Inhibition of Methylisocitrate Lyase Activity by Oxidation-reduction Coenzymes as a Function of Methylisocitrate Concentration.

•, none; A, NADPH (1 mm); E1, NADH (1 mm); x, NADP (1 mm); 0, NAD (1 mm); G, NAD (2 mm).

caused changes in imax of methylisocitrate lyase.

DISCUSSION

Methylisocitrate lyase requires divalent metal ions such as Mg2+ and a sulfhydryl compound

such as cysteine in the assay buffer in order to show full activity and is inhibited by the

sulfhydryl reagents, as shown in all isocitrate .16•`18)

The reaction catalyzed by methylisocitrate lyase is a reversed aldol condensation, similar

FIG. 5. Effect of Various Coenzymeson Enzyme to that catalyzed by isocitrate lyase. ISO- Activity. citrate lyase had been known to have a narrow Coenzymeswere added at a concentrationof 1 mm. substrate specificity and to cleave only threo-

Ds-isocitrate, but McFadden et al.19) have The effect of several coenzymes on the en described that several bacterial and fungal zyme activity was examined (Fig. 5). Al isocitrate lyases are able to cleave MICA at a though AMP, ADP, and ATP did not seem to variety of rates between 2 and 20 % of that of affect the enzyme activity, NADH and NAD- isocitrate cleavage. The preceding paper" has PH appreciably inhibited it. On the contrary, described that the isocitrate lyase from C. NAD showed a stimulatory effect. The in lipolytica is able to cleave MICA at a rate of hibition by NADH and NADPH was non- about 3 % of that of isocitrate cleavage. competitive with methylisocitrate (Fig. 6). MICA is the only substrate known to be These effectors, NADH, NADPH and NAD, cleaved by methylisocitrate lyase: any of 174 T. TABUCHI and T. SATOH

citrate, malate, and threo-Ds and threo-DL cells might inhibit some part of reactions isocitrates are inert. On the contrary, methyl- which are located at the earlier stages of the citrate synthase, the companion enzyme of the cycle and lead to further production of pro methylcitric acid cycle, is found to have a pionyl-CoA, although interpretations of broad substrate specificity for acyl-CoA deriva studies in vitro should be extrapolated to tives. 8' This broad substrate specificity re phenomena in vivo with reservation. sembles that of acetyl-CoA carboxylase (EC 6.4.1.2, acetyl-CoA: carbon-dioxide Acknowledgment. This work was supported in part by a scientific grant from the Ministry of Education (ADP)).'" The usual citrate synthase of C. of Japan. lipolytica can not catalyze methylcitrate synthesis.') The isocitrate lyases from Pseudomonas REFERENCES indigofera2l'22) and Neurospora crassa23) are 1) T. Tabuchi, N. Serizawa and H. Uchiyama, Agric. reported to be inhibited by fumarate, succinate, Biol. Chem., 38, 2571 (1974). malate, glycolate, oxalate, malonate, itaconate, 2) T. Tabuchi and N. Serizawa, ibid., 39,1055 (1975). 3) T. Tabuchi and S. Hara, ibid., 38, 1105 (1974). or phosphoenolpyruvate. Any of these acids 4) T. Tabuchi and S. Hara, Nippon Nogeikagaku has little or no effect on the methylisocitrate Kaishi, 48, 543, 549 (1974). lyase from C. lipolytica. The enzyme, however, 5) T. Tabuchi, N. Serizawa and S. Ohmomo, Agric. is inhibited markedly by NADH and NADPH, Biol. Chem., 38, 2565 (1974). and activated by NAD. The first enzyme in the 6) T. Tabuchi and N. Serizawa, ibid., 39,1049 (1975). methylcitric acid cycle, methylcitrate synthase, 7) T. Tabuchi and H. Uchiyama, ibid., 39, 2035 (1975). is also inhibited by NADH, NADPH, and 8) H. Uchiyama and T. Tabuchi, ibid., 40, 1407 ATP. (1976). As shown in Fig. 1, the net effect of one 9) T. Tabuchi and T. Satoh, ibid., 40, 1863 (1976). turn of the cycle is propionyl-CoApyruvate 10) O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. L. Randall, J. Biol. Chem., 193, 265 (1951). +CoASH+4H; this formula can be modified 11) B. J. Davis, Ann. N. Y. Acad. Sci., 121,404 (1967). as follows: propionyl-CoA+O2+5ADP+5P1 12) A. Kornberg and W. E. Price, Jr, J. Biol. Chem., pyruvate+CoASH+ 5ATP. The operation 189, 123 (1951). of the methylcitric acid cycle means the pro 13) C.J.R. Thorne, L.I. Grossman and N.O. Kaplan, duction of energy in cellular metabolism. Biochim. Biophys. Acta, 73, 193 (1963). These experimental results support the idea 14) E. Racker, ibid., 4, 211 (1950). 15) G. H. Dixon and H. L. Kornberg, Biochem. J., that the predominant physiological role of 72, 3P (1959). the methylcitric acid cycle is not a supply of 16) B. A. McFadden and W. Howes, J. Biol. Chem., biosynthetic intermediates related to pro 238, 1737 (1963), pionate but a purely catabolic reaction se 17) I. Shiio, T. Shiio and B. A. McFadden, Biochim. Biophys. Acta, 96, 123 (1965). quence concerning the oxidation of propionate 18) J. T. McCarthy and A. M. Charles into pyruvate and that both methylcitrate , Can. J. Microbiol., 19, 513 (1973). synthase and methylisocitrate lyase are the 19) B. A. McFadden, I. A. Rose and J . O. Williams, regulating of this cycle. Arch. Biochem. Biophys., 148, 84 (1972). It is well known that the intracellular con 20) M. Waite and S. J. Wakil, J. Biol. Chem., 237, centrations of NAD(P)H and/or ATP serve to 2750 (1962). regulate the further production of these sub- 21) G. R. Rao and B. A. McFadden , Arch. Biochem. Biophys., 112, 294 (1965). stances. Such regulation might well be exerted 22) J. O. Williams, T. E. Roche and B. A . McFadden, on the activities of methylcitrate synthase and Biochemistry, 10, 1384 (1971). methylisocitrate lyase for fine control of the 23) R. A. Johanson, J. M. Hill and B. A. McFadden, ratio of oxidation of propionyl-CoA, and an Biochim. Biophys. Acta, 157, 327 (1974) . elevated concentration of propionyl-CoA in