Agr. Biol. Chem., 40 (9), 1863•`1869, 1976

Distinction between Isocitrate and in 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 May 6, 1976

Examination of DEAE-cellulose chromatography clearly demonstrated the presence of the cleaving 2-methylisocitrate into pyruvate plus succinate in the methylcitric acid cycle. The enzyme differed from the usual of Candida lipolytica, and was herein given the trivial name of methylisocitrate lyase. The chromatography also proved the mutant strain of C. lipolytica, producing 2- methylisocitrate from odd-carbon n-alkanes, to lack this methylisocitrate lyase. The presence of isozyme of isocitrate lyase was not detected in C. lipolytica. The isocitrate lyase of C. lipolytica could cleave 2-methylisocitrate at a rate of 3 % of that of isocitrate cleavage. On the contrary, methylisocitrate lyase hardly acted on isocitrate. Two differed in chromatographic elution patterns, pH optima, kinetics of thermal inactiva tion, and behavior with inhibitors, such as itaconate and , in addition to specificity. The experimental results revealed that methylisocitrate lyase is impossible to be the isozyme of isocitrate lyase, and justified the previously proposed ideas that propionyl- CoA is oxidized through the methylcitric acid cycle in C, lipolytica and that the absence of this enzyme in the mutant strain caused the extracellular accumulation of 2-methylisocitrate from odd-carbon n-alkanes.

Previously we proposed the methylcitric acid on substrate specificity of isocitrate lyase cycle concerning the oxidation of propionyl- (EC 4.1.3.1), that fungal and bacterial isocitrate CoA into pyruvate via seven-carbon tricar- lyases cleaved MICA to equimolar pyruvate boxylic acids (Fig. 1)1,2) on the basis of the and succinate although the rates of cleavage finding of the accumulation of threo-Ds-2- of MICA by these lyases were considerably methylisocitric acid (MICA) in large amounts lower than those for threo-Ds from odd-carbon n-alkanes by a mutant (ICA); this cleavage of MICA is the same strain of Candida lipolytica, No. R-2, and reaction as involved in the present cycle (III in other observations.1•`7) Enzymatic evidence Fig. 1). As previously described,7)the activity was presented that the cell-free extracts of of MICA cleavage by the cell-free extract of C. lipolytica could catalyze the three key the mutant strain of C. lipolytica was about reactions involved in this cycle (I, II, and III one-twentieth of that of the parent strain, in Fig. 1).1,7) In the preceding paper," methyl- although the activity of ICA cleavage by citrate synthase, the key enzyme located at the the extract of the mutant strain was about entrance of this cycle, was purified and shown three times as high as that of the parent strain: to differ from the usual citrate synthase (EC these experimental results suggested that the 4.1.3.7) of C. lipolytica. usual isocitrate lyase of C. lipolytica could

McFadden et al.9) had reported, in a study not play a substantial role in MICA cleavage and that MICA was cleaved exclusively by an Abbreviations: MICA, threo-Ds-2-methylisocitric additional enzyme being absent or poor in acid; ICA, threo-Ds-isocitric acid; TMM buffer, 0.01 m the mutant strain. Tris-HC1 buffer, pH 7.5, containing 3 mm MgCl2 and 1 mm 2-mercaptoethanol. The multiforms of isocitrate lyase have 1864 T. TABUCHI and T. SATOH

, 100g; Polypeptone, 10g; KH2PO4, 0-5g; MgSO4.7H20, 0.5 g; thiamine-HCI, 50 ƒÊg bromocresol

purple, 0.02g, in tap water. The n-alkanes medium contained per liter: a mixture of n-alkanes (C12•`C15),

30 ml, as carbon source; Span 80, 0.2 g; and the other ingredients were the same as shown in the glucose

medium.

Growth conditions. To prepare seed culture, cells

grown on potato-glucose agar slant were inoculated into 50 ml of the medium contained in a 500-m1

Erlenmeyer flask, and incubated for 2 days at 26°C on a rotary shaker at 220 rpm. This culture (2 ml)

was transferred into 50 ml of the same medium con tained in 500-m1 Erlenmeyer flasks. Cells were grown

under the same conditions as for the seed culture except that 20%. sodium carbonate solution was added

dropwise into each flask three times a day to keep

the pH of the culture at about 4.5 to 6.5 according to change in color indicated by bromocresol purple.

After incubation for 3 days, cells were harvested by

centrifugation, washed twice with 0.01 m Tris-HCl buffer at pH 7.5, and stored at -20•Ž.

FIG. 1. The Stages of the Methylisocitric Acid Preparation of cell-free extracts. The cells were Cycle. suspended in two to three times their weight of 0.01 M

Tris-HCI buffer, pH 7.5, containing 3 mm MgC12 and recently been reported to exist in Neurospora 1 mm 2-mercaptoethanol (hereafter referred to as TMM

crassa,10,11) one of the Pseudomonas species,12) buffer), and disrupted three times with an X-press at and .13•`15) The possibility, then, 4•Ž. remained unclear that an isozyme of isocitrate Enzyme assays. The composition of reaction lyase in C. lipolytica might cleave MICA. mixture was similar to that described by Dixon and

This paper deals with the chromatographic Kornberg:16) the sodium salt of ICA or MICA, 2.5

separation of the capable of cleaving ,ƒÊmoles, as substrate; MgCl2, 25,ƒÊmoles; phenylhyd-

ICA or MICA and with preliminary examina razine-HCI, 10 ymoles; cysteine-HCI 18 ,ƒÊmoles; tion on their properties. The enzyme cleaving phosphate buffer at pH 6.5, 600 ƒÊmoles, for ICA cleavage, or Tris-HCI buffer at pH 7.5, 600 ƒÊmoles, MICA was clearly separated from the iso for MICA cleavage, (unless otherwise specified); and citrate lyase of C. lipolytica by column enzyme in a final volume of 2.5 ml. The change in chromatography; the former could hardly absorbance at 324 nm due to glyoxylate or pyruvate

cleave ICA but the latter could weakly cleave phenylhydrazone was followed continuously on a MICA. The presence of isozyme of iso Hitachi, Model 124, spectrophotometer equipped with a recorder. The substrate was finally added after citrate lyase was not detected in C. lipolytica. temperature equilibrium, 25•Ž, had been established and The enzyme cleaving MICA was completely a baseline had been run on the recorder. The molar lost in the mutant strain. The trivial name extinction coefficient of glyoxylate phenylhydrazone17)

of methylisocitrate lyase will be given here- was taken to be 16.8 mm-1 cm-1 and that of after to this enzyme which was proved to pyruvate phenylhydrazone was calculated from the absorption spectrum of this compound to be 10.2 mm-1 differ from isocitrate lyase. cm-1,.

Protein was determined by the method of Lowry MATERIALS AND METHODS et al.18) One unit of enzyme activity is defined as that amount which produces one nanomole of glyoxylate or

Microorganisms. C. lipolytica IFO 1659 and its pyruvate from ICA or MICA per min at 25•Ž. mutant strain, No. R-2,3,4) were used. Specific activity is expressed as units per mg of protein.

Media. The glucose medium contained per liter: DEAE-cellulose chromatography. Cell-free extracts Isocitrate Lyase and Methylisocitrate Lyas 1865

were passed through DEAE-cellulose columns (30 by

2.0 cm) previously equilibrated with TMM buffer . The columns were washed with 500 ml of TMM buffer.

Enzymes were eluted at a flow rate of about 100 g

per hour with a Tris-HC1 gradient between 200 ml of the original TMM buffer and 200 ml of a modified

TMM buffer containing Tris-HCl of the same pH at

a concentration of 0.2 M instead of the original 0.01 M, unless otherwise specified. Fractions (3 g)

were collected and assayed for the activities of iso

citrate- and methylisocitrate-cleavages. The active fractions were pooled separately and examined for

several properties.

Sephadex G-200 gel filtration. Portions of both

enzyme preparations obtained by DEAF-cellulose

chromatography were combined and concentrated with a collodion bag apparatus. The concentrate was

placed on a Sephadex G-200 column (100 by 2.4 cm)

previously equilibrated with TMM buffer. The enzy mes were eluted with TMM buffer at a flow rate of

30 g per hour and fractions (5 g) were collected. Isocitrate- and methylisocitrate-lyases were located in

the fractions by assaying each tube for enzyme activity.

Thermal inactivation. Both preparations of iso

citrate- and methylisocitrate-lyases obtained by DEAE- cellulose chromatography were incubated with TMM FiG. 2. DEAF-Cellulose Column Chromatography

buffer at 50•Ž. Samples (0.05 ml) were withdrawn of Extracts Obtained from Cells Grown on n-Alkanes. and assayed for enzyme activity at the indicated time (A) The Parent Strain, (B) the Mutant Strain.

intervals (Fig. 6). •›-•›, the activity of isocitrate cleavage; •œ-•œ , the activity of 2-methylisocitrate cleavage; -•E-•E- , Tris- Identification of reaction products. The products of HCl concentration. reactions of isocitrate- and methylisocitrate-lyases were identified by paper- and gas-chromatography.6,7) and the latter to methylisocitrate lyase,

Chemicals. ICA19) and MICA3,4) used as sub- respectively, on the basis of their substrate strates were the products isolated from culture broths specificity. of C. lipolytica, and they were proved gas-chromato- The cleavage products by the isocitrate- graphically',') not to be contaminated by each other. lyase fractions were confirmed to be succinate All other chemicals were obtained as described pre- plus glyoxylate and those by methylisocitrate- viously.,,,) lyase fractions to be succinate plus pyruvate, respectively, by paper- and gas-chromato- RESULTS graphy. Elution patterns of isocitrate- and methyl- Clearly, the peak fractions of isocitrate

isocitrate-lyases from DEAE-cellulose colu- lyase showed the activity of MICA cleavage mns at a rate of about 3 % of that of ICA cleavage. Figure 2 shows the results of DEAE-cellulose MICA used as substrate was proved to con chromatography of enzyme activity in the tain no detectable amounts of isocitrate by alkane-grown cells of both strains. In the gas-chromatography. parent cells (Fig. 2A), two main peaks of On the contrary, hardly any activity of enzyme activity were distinctly separated: one ICA cleavage was detected at the peak frac eluted in the 0.075 M Tris-HCl fraction and tions of methylisocitrate lyase. the other in the 0.11 M Tris-HCJ fraction. As Fig. 2B shows, no fraction containing The former corresponded to isocitrate lyase methylisocitrate lyase was observed in the 1866 T. TASucui and T. SATOH

mutant cells grown on n-alkanes. The pos These chromatographic patterns were high- sibility was excluded that any activity of ly reproducible in both strains but the presence methylisocitrate cleavage remained uneluted, of isozyme of isocitrate lyase was not detected because the initial activity of the extract in in every cases. extremely small amounts was almost recovered in the fractions corresponding to isocitrate Elution patterns of isocitrate- and methyl lyase. These results clearly indicate that the isocitrate-lyases in Sephadex G-200 gel mutant cells grown on n-alkanes lost methyl- filtration isocitrate lyase. As Fig. 4 shows, the distribution curves of Figure 3 shows the results of DEAE-cellulose elution of the two enzymes deviated slightly chromatography of enzyme activity in the from each other in Sephadex G-200 gel glucose-grown cells of both strains. The filtration; isocitrate lyase was eluted earlier. parent cells grown on glucose (Fig. 3A) clearly These patterns imply that the molecular gave the two peaks of enzyme activity cor- weight of isocitrate lyase is larger than that responding to isocitrate- and methylisocitrate- of methylisocitrate lyase, if both enzymes are lyases, although both peaks, especially the spherical. one corresponding to isocitrate lyase were low as compared with those grown on n-alkanes. The mutant cells grown on glucose (Fig. 3B) also lost methylisocitrate lyase.

FIG. 4. Sephadex G-200 Column Chromatography

of Isocitrate- and Methylisocitrate-lyases.•›•\•›

, the activity of isocitrate cleavage; •œ•\•œ, the activity of methylisocitrate cleavage.

Effect of pH on enzyme activity The effect of pH on both enzyme activities was examined by the standard assay method except that the pH of reaction was varied. As Fig. 5 shows, the optimum pH for isocitrate lyase was observed between 6.5 and 6.8, and that for methylisocitrate lyase between 7.3 FIG. 3. DEAF-Cellulose Column Chromatography and 7.8. From these observations, the phos of Extracts Obtained from Cells Grown on Glucose . phate buffer of pH 6.7 and the Tris-HCI (A) The Parent Strain, (B) the Mutant Strain. buffer of pH 7.5 were employed in the standard •›•\•›, the activity of isocitrate cleavage; •œ•\•œ, the activity of 2-methylisocitrate cleavage; -•E-•E- assay mixture for isocitrate lyase and in that , Tris-IIC1 concentration. for methylisocitrate lyase, respectively . Isocitrate Lyase and Methylisocitrate Lyase 1867

TABLE I. EFFECT OF ITACONATE AND OXALATE ON ACTIVITIES OF ISOCITRATE- AND METHYLLSOCITRATE-LYASES

FIG. 5. Effect of pH on Activities of Isocitrate- conate and oxalate behaved as strong inhibitors and Methylisocitrate-lyases. against the isocitrate lyase of C. lipolytica,

•›, isocitrate lyase activity; •œ, methylisocitrate lyase similarly as reported in Pseudomonas indigo activity; ---, phosphate buffer; -, Tris-HCl buffer. fera.20) In striking contrast, itaconate and oxalate did not inhibit the activity of methyl Thermal inactivation of isocitrate- and methyl isocitrate lyase. isocitrate-lyases The inhibitory effect of MICA on ICA The rates of thermal inactivation of both cleavage by isocitrate lyase was not detected enzymes was examined at 50•Ž. Figure 6 within the range of concentrations of MICA is the plot of logarithm of percentages of tested (0.6 to 2.4 mm). In addition, the in remaining activities versus time. Isocitrate hibitory effect of ICA on MICA cleavage by lyase was extremely sensitive compared with methylisocitrate lyase was also not detected. methylisocitrate lyase. The remaining acti

vities of ICA-cleavage paralleled fairly well DISCUSSION to those for MICA-cleavage by isocitrate

lyase: this finding indicated that the isocitrate The enzymes, isocitrate- and methylisocit-

lyase preparation used was not contaminated rate-lyases, catalyze similar reactions in ICA by methylisocitrate lyase. cleavage and in MICA cleavage, respectively. The isozymes of isocitrate lyase have been re-

ported to exist in several ;10•`15' they were separated from DEAE-cellulose columns at different salt concentrations. On

the other hand, McFadden et al.9) reported that maximal velocity ratios for ICA/MICA

cleavage by isocitrate lyases varied from 5 to 50 among several bacterial and fungal

enzymes. The considerable difference in the

ratios might be explained on the assumption that the velocity ratios differed between the

isozymes of isocitrate lyase and the propor-

tions of the isozymes also differed among the FIG. 6. Kinetics of Thermal Inactivation at 50•Ž.•›•\•› fungal and bacterial enzymes. Clearly, the , isocitrate lyase activity; •œ•\•œ, methylisocitrate lyase activity. present results indicate that the enzyme cleav- ing MICA, methylisocitrate lyase, differs from Inhibition studies isocitrate lyase, although the presence of The effect of several compounds on enzyme isozymes of isocitrate lyase was not detected in activity was tested. As Table I shows, ita- C. lipolytica. 1868 T. TABUCHiand T. SATOH

The isocitrate lyases of a , Tur pared with the parent strain, in odd-carbon batrix aceti, were reported to consist of five n-alkanes media containing no neutralyzing isozymes.13) In addition, the nematode, Neu agent:2) in these cultures, isocitrate lyase rospora and Pseudomonas enzymes all were having the weak activity of MICA cleavage reported to consist of four subunits."' The might function in MICA cleavage which is molecular weight of methylisocitrate lyase necessary to metabolize propionyl-CoA was inferred to be smaller than that of the through the methyleitrate cycle if the mutant isocitrate lyase of C. lipolytica from the strain has not any other pathway than the observation on patterns of gel filtration, if methylcitric acid cycle. both enzymes were spherical. The possibility Both lyases also differed in pH optima, that methylisocitrate lyase, the smaller protein, kinetics of thermal inactivation, and behavior is related to isocitrate lyase as a protomer is with itaconate and oxalate. More detailed improbable, because the mutant strain could properties of methylisocitrate lyase will be synthesize isocitrate lyase but not methyliso described in the next paper. citrate lyase. Previously, we suggested that an additional Acknowledgment. This investigation was supported in part by a grant from the Ministry of Education of enzyme other than the usual isocitrate lyase Japan. of C. lipolytica cleaved MICA and that the enzyme was absent or poor in the mutant strain.7) The present results indicate that REFERENCES the mutant strain completely lacked methyl 1) T. Tabuchi, N. Serizawa and H. Ucbiyama, isocitrate lyase: this finding justifies the pre- Agr. Biol. Chem., 38, 2571 (1974). viously proposed ideas that propionyl-CoA is 2) T. Tabuchi and N. Serizawa, ibid., 39, 1055 oxidized through the methylcitric acid cycle in (1975). the parent strain and that the absence of 3) T. Tabuchi and S. Hara, ibid., 38, 1105 (1974). 4) T. Tabuchi and S. Hara, Nippon Nogeikagaku this enzyme in the mutant strain causes the Kaishi, 48, 549 (1974). accumulation of MICA from odd-carbon 5) T. Tabuchi, N. Serizawa and S. Ohmomo, Agr. n-alkanes. Biol. Chem., 38, 2565 (1974). The isocitrate lyase of C. lipolytica was 6) T. Tabuchi and N. Serizawa, ibid., 39, 1049 found to be capable of cleaving MICA at a (1975). 7) T. Tabuchi and H. Uchiyama, ibid., 39, 2035 rate of 3 % of that of ICA cleavage under the (1975). standard assay conditions. On the contrary, 8) H. Uchiyama and T. Tabuchi, ibid., 40, 1411 ICA cleavage by methylisocitrate lyase was (1976). hardly discernible. The cell-free extract of 9) B. A. McFadden, I. A. Rose and J. O. Williams, the mutant strain lacking methylisocitrate Arch. Biochem. Biophys., 148, 84 (1972). lyase showed the weak activity of MICA 10) R. E. Sjorgen and A. H. Romano, J. Bacteriol., cleavage;7) this cleavage can be accounted 93, 1638 (1967). 11) R. B. Flavell and D. O. Woodward, ibid., 105, for by the weak action of isocitrate lyase. 200 (1971). The mutant strain produced extracellularly 12) E. Bellion and J. Woodson, ibid., 122, 557 (1975). MICA in large amounts when incubated in 13) U. Reiss and M. Rothstein, Biochemistry, 13, odd-carbon n-alkanes media containing a 1796 (1974). 14) U. Reiss and M. Rothstein, Biochem. Biophys. neutralizing agent;3,6) this phenomenon in the Res. Commun., 61, 1012 (1974). mutant strain might imply the detoxication of 15) W. J. Colonna and B. A. McFadden , Arch. propionyl-CoA derived from the alkanes. On Biochem. Biophys., 170, 608 (1975). the other hand, in media containing no 16) G. H. Dixon and H. L. Kornberg, Biochem. J., neutralyzing agents, acid production by C. 72, 3P (1959). 17) G. Turian and M. Kobr, Biochim. Biophys . Acta, lipolytica was depressed as a rule.21) The 99, 178 (1965). mutant strain grew extremely slowly, as com 18) O. H. Lowry, N. J. Rosebrough, A. L. Farr and Isocitrate Lyase and Methylisocitrate Lyase 1869

R. J. L. Randall, J. Biol. Chem., 193, 265 (1951). 20) G. R. Rao and B. A. McFadden, Arch. Biochem. 19) M. Abe and T. Tabuchi, Agr. Biol. Chem., 32, Biophys., 112, 294 (1965). 392 (1968); M. Abe, T. Tabuchi and M. Tanaka, 21) T. Tabuchi, Nippon Nogeikagaku Kaishi, 47, 479 Nippon Nogeikagaku Kaishi, 44, 493 (1970). (1973).