Purification and Someproperties of Arginine Deiminase in Euglena

Agric. Biol. Chem., 48 (2), 483-489, 1984 483

Purification and SomeProperties of Arginine Deiminase in Euglena gracilis Z Bong-Sun Park, Aiko Hirotani, Yoshihisa Nakano and Shozaburo Kitaoka Department of Agricultural Chemistry, University of Osaka Prefecture, Sakai, Osaka 591, Japan Received August 29, 1983

In Euglena gracilis arginine deiminase was located in the mitochondrial matrix. The highly purified enzyme required Co2+for the enzyme reaction with the Kmvalue of 0.23mM, and its optimum pH was 9.7 to 10.3. The molecular weight of the native enzyme protein was 87,000 by gel filtration, and SDS-acrylamide gel electrophoresis showed that the enzyme consisted of two identical subunits with a molecular weight of 48,000. Euglena arginine deiminase was inhibited by sulfhydryl inhibitors, indicating that a sulfhydryl group is involved in the active center of the enzyme. It exhibited negative cooperativity in binding with arginine. L-a-amino-jS-guanidinopropionate, D-arginine, and L-homoarginine strongly inhibited the enzyme while /?-guanidinopropionate, y-guanidinobutyrate, and guanidinosuccinate did not. Considerable inhibition was also observed with citrulline and ornithine. Wediscuss the effects of the unique properties of the Euglena arginine deiminase on the regulation of arginine metabolism in this protozoon.

Euglena gracilis accumulates arginine as the MATERIALS AND METHODS major nitrogen reserve depending on the phase Organism and culture. E. gracilis strain Z was grown of growth whena relatively excess of a ni- under illumination (2500lux) at 27°C as described trogen source is supplied, and upon acquiring previously. 1} a carbon source the amino acid is rapidly me- tabolized for renewed cell growth.1} Arginine Fractionation of cell homogenate. Acell homogenate was as a cellular nitrogen reserve has been report- obtained by partial trypsin digestion of the pellicle fol- ed in higher plants2) and Neurospora.3) In lowed by mild mechanical disruption,10) and fractionated by differential centrifugation according to Shigeoka et Euglena arginine is catabolized to ornithine al.n) via citrulline by the action of arginine deiminase and citrullinase, or by the so-called Preparation of crude enzyme. A culture of E. gracilis in arginine dihydrolase pathway. 1} the late-exponential phase of growth (107 cells/ml) was supplemented with 200 mMethanol (final concentration), Manyreports have dealt with the occurrence and after 12 hr of incubation it was centrifuged at 2000 x g of arginine deiminase in prokaryotes4~6) and for 5min to harvest cells. The cells (24g) were washed eukaryotes7~9) and there are a few reports on twice with distilled water, suspended in 10ml of 10mM the purification of this enzymein bacteria,5>6) potassium phosphate buffer, pH 7.0, and disrupted by but no information is available on the purifi- sonication (10 kc) for a total of 3min with ll intervals of 15 sec each. The supernatant obtained by centrifugation of cation and detailed properties of the enzyme the sonicate at 9000xgfor 15minwas used as a crude in eukaryotic organisms. In the present paper enzyme.The typical preparation showed an enzymeac- the purification and some properties of ar- tivity of 0.38 fimol of citrulline formed per mg protein per ginine deiminase in E. gracilis are described min, whenthe arginine concentration was 50mM. and the significance of the enzyme in the Protein was determined by the method of Bradford.12) regulation of arginine metabolism in Euglena is discussed. Enzyme assays. Arginine deiminase was assayed by measuring labeled citrulline formed from [U-14C]arginine 484 B.-S. Park et al.

(0.1 juCi/mM) by the enzyme reaction as described pre- Electrophoresis was carried out at a constant current viously.1} Succinate semialdehyde dehydrogenase, a mi- (5 mA/gel) with bromophenol blue as a migration mark- tochondrial marker enzyme;13) glucose 6-phosphatase, a er. Proteins in the gel were stained with Coomassie bril- microsomal marker enzyme;14) ribulose- l ,5-bisphosphate liant blue R-250 and destained in 7% acetic acid. carboxylase, a chloroplast-marker enzyme;15) and glutamate dehydrogenase, a cytosolic marker enzyme10)were RESULTS assayed by the respectively cited methods. Subcellular location of arginine deiminase Molecular weight determination by gel filtration. The Distribution of arginine deiminase and molecular weight of the enzymewas determined by the marker enzymes in the subcellular fractions method of Andrews16* using a column (1.5x60cm) of Sephacryl S-200. Chromatography was carried out at of E. gracilis is shown in Table I. The deimin- 4°C at a flow rate of 30ml per hr using 100him potas- ase was located in the mitochondrial fraction, sium phosphate buffer, pH 7.4, as an eluant. The column as judged from the distribution of activities was calibrated with lactate dehydrogenase (pig heart), of marker enzymes. Sonication of the iso- malate dehydrogenase (pig heart), trypsin inhibitor lated mitochondrial fraction released the (soybean), and cytochrome c (horse heart). deiminase as well as succinate semialdehyde SDS-Polyacrylamide gel electrophoresis. SDS-Poly- dehydrogenase into the 100,000 x g superna- acrylamide gel electrophoresis of the purified arginine tant. The slight amount of the deiminase in deiminase was performed according to Weber and the chloroplast fraction was due to con- Osborn.17) The enzyme and known protein standards (Electrophoresis calibration kit, Pharmacia Fine tamination by mitochondria. Chemicals) were treated by heating for 5 min at 100°C in a solvent system consisting of 1%SDS, 1% 2-mercapto- Purification of arginine deiminase ethanol and 10mM sodium phosphate buffer, pH 7.2. Typical purification steps of the Euglena

Table I. Distribution of Arginine Deiminase and Marker Enzymes IN SUBCELLULAR FRACTIONS OF E. gracilis

Enzymeactivity0

Cytosol Mitochondria Microsomes Chloroplasts

Arginine deiminase 7.4 78.0 3.9 1 3.6 (0.03)fc (1.53)* (0. 13)b (0.51)b Glutamate dehydrogenase (NADP +) 94. 1 0 0 0 Succinate semialdehyde dehydrogenase (NADP+) 5. 1 86.9 0 3.5 Glucose 6-phosphatase 0 0 27.3 0 Ribulose- 1 , 5-bisphosphate carboxylase 7.5 9.9 3.7 76.0

%against the activity in the crude extract. Specific activity, /miol/mg protein/min.

Table II. Purification Steps of Euglena Arginine Deiminase Total protein Specific activity Total activity Recovery ^ (mg) (jumol/mg protein/min) (jumol/min) ( %) Crude extract 1202 0.0284 14. 14 100 Ultracentrifugation 584.0 0.0345 20. 1 5. 59 DEAE-Cellulose 35.75 0.466 16.66 49 P-Cellulose 1 1.38 1.38 15.74 46 Sephacryl S-200 5.04 1.59 8.03 24 Arginine deiminase in Euglena gracilis 485 arginine deiminase are summarized in Table II. Determination of molecular weight The crude enzyme was centrifuged at Figure 2 shows plots of the elution volume 105,000xg for 60min with a Hitachi 65P from a Sephacryl S-200 column against log- ultracentrifuge, and the supernatant was ap- arithms of the molecular weight of the standard plied onto a DEAE-cellulose column (3 x proteins and arginine deiminase. The molec- 20cm) equilibrated with 10mM potassium ular weight of this enzymewas calculated to phosphate buffer, pH 7.0. After washing with be about 87,000. SDS-polyacrylamide gel 50 ml of the same buffer the columnwas eluted electrophoresis of the deiminase together with a linear concentration gradient (0 ~0.3 m) with several proteins with knownmolecular of potassium chloride and active fractions weight indicated that the subunit of this en- were brought to 70%saturation of ammonium zyme had a molecular weight of 48,000, as sulfate. The precipitate was dissolved in the shown in Fig. 3. above buffer and passed through a Sephadex G-25 column (2.5 x45cm) to remove the am- Effects of temperature and pH moniumsulfate. The protein fraction was ap- This enzyme was stable up to 40°C after plied onto a column (2 x 15cm) of P-cellulose treatment for 15 min, and retained 61.2% of its equilibrated with 10 mMpotassium phosphate activity after treatment at 50°C (Fig. 4). buffer, pH 7.4, and the column was eluted with a 10 to 200mMgradient of potassium phos- 20r phate buffer, pH 7.0. The unadsorbed, active - \A fractions were precipitated by the addition of 10- \ ammoniumsulfate to 70% saturation, and the precipitate was dissolved in 50 mMphosphate buffer, pH 7.0. The enzyme solution was ap- plied onto a Sephacryl S-200 column (2.5 x

55cm) and eluted with the 50mMbuffer. The 110 .20 30 elution pattern (Fig. 1) showed only one peak Fraction number of deiminase activity, and the active fractions (Nos. 18 through 27) were combined and Fig. 2. Molecular Weight Determination by Gel Filtration. stored at -20°C. The enzyme preparation A Sephacryl S-200 column was calibrated with known thus obtained had been purified 56-fold over protein standards, including lactate dehydrogenase the crude enzyme, giving 24%overall recovery (1 35,000) (A), malate dehydrogenase (70,000) (C), Trypsin of the activity. The preparation exhibited inhibitor (20,100) (D), and cytochrome c (12,400) (E). (B), only one detectable protein band in SDS- Euglena arginine deiminase. polyacrylamide disc gel electrophoresis. J Op-ox 0A" -3t |5: >&

I 0-2,1-i-i-j- 0-6 1-0 Migration ratio Fig. 3. Molecular Weight Estimation by SDS-Acryl- amide Gel Electrophoresis. u 0 20 AO 60 A, phosphorylase b (94,000); B, bovine serum albumin Fraction number (67,000); C, Euglena arginine deiminase; D, ovalbumin Fig. 1. Elution Pattern by Sephacryl S-200 Gel (43,000); E, carbonic anhydrase (30,000); F, soybean Filtration. trypsin inhibitor (20,100); G, a-lactalbumin (14,400). 486 B.-S. Park et al.

Table III. Requirement of Metal Ions 110- °~~2^V\ for EnzymeActivity Addition Enzyme activity ( 1 him) (jumol/mg protein/min) fO* / \Q 50-5- \ None 0. 12 CoCl2 1.83 MgCl2 0.78 MnCl2 0. 12 CaCl2 0. 15 o Lj 1 i u NiCl2 0. 14 0 20 40 60 Temperature (°C ) FeSO4 0. 14 CoCl2 +EDTA 0.15 Fig. 4. Effect of Temperature on Arginine Deiminase. For determining temperature optimum (#) enzyme was assayed under standard conditions but at various temperatures, and for temperature stability (O) enzyme was kept !2~ ^ ~-o for 15 minat varied temperatures before assaying. I / 2r ^

<3 X °0 10 i/ic

o L-l0 1 0-5 1 1 lJ1-0 C0CI2 concentration (mM) ifT\|0-5- Fig. 6. Effect of CoCl2 Concentration on Arginine i-f Deiminase.

ot: 1 1 Table IV. Effects of Sulfhydryl Reagents 8 9 10 ll PH Fig. 5. pH Optimumof Arginine Deiminase. Enzyme ...... activity Inhibition Enzymewas assayed in 50dim each of potassium phos- A ddition0 .. à" phate buffer (O), glycine-KOH buffer (0) or sodium Gimol/mg ( %) bicarbonate buffer (A) under standard assay conditions protein/min) but at different pHs. None 1.19 0

/7-Chloromecuribenzoate,/ri. i \ u.ll yu.o Figures 4 and 5 show that the maximum 5,5 /-Dithiobis(2-nitrobenzoate) 0. 28 86. 5 activity of the enzyme is obtained at 30°C and A^-Ethylmaleimide(0.10. 8mM)3 30. 3 pH 9.7-10.3. The enzyme was stable in the HgCl2 0.33 72.3 range of pH8.0 to 1 1.0 when treated at various Mersaryl 0.22 81.5 pHs for 15min at 40°C before the enzyme Mersaryl(2him) + 2-mercaptoethanol reaction. Isonicotinic acid hydrazide 1.25 - 5.0 Aminoxyacetate 1. 14 4.2 Effects of metal ions and some compounds Semicarbazide 1. 1 8 0.8 As shown in Table III, Co2+ was essential to the enzymeactivity, and Mg2+could replace At 1 him unless otherwise specified. Co2+ with lower activity. Other metal ions tested hardly affected the enzyme activity. The Euglena arginine deiminase was inhibited activation of the enzyme by Co2+ was com- 77-82% by sulfhydryl inhibitors like p- pletely removed by EDTA. Figure 6 shows chloromercuribenzoate, 5,5'-dithiobis-(2- that 0.5 mMCoCl2 gives the maximumenzyme nitrobenzoate), JV-ethylmaleimide, Hg2 +, and activity, and that the Kmvalue is 0.23him. mersaryl, and the enzyme was protected Arginine deiminase in Euglena gracilis 487 Effects of substrate concentration and arginine 20 - O^ analogues Figure 7 shows that the activity of arginine 5 / deiminase exhibits triphasic proportion when "oo. r p gMO- / the arginine concentration wasvaried, indicating that the enzyme has plural combining sites with different affinities for arginine. Effects of some L-arginine analogues con-

q QL I \ I i taining guanidino groups and nitrogenous 0 20 40 Arginine concentration (mM) compoundsrelated to arginine metabolism on the enzymatic hydrolysis of arginine are shown Fig. 7. Effect of Arginine Concentration on Enzyme Activity. in Table V. L-a-amino-/}-guanidinopropionate, D-arginine, and L-homoarginine strongly inhibited the hydrolysis while jS-guanidinopro- Table V. Effects of Arginine Analogues pionate, y-guanidinobutyrate and guanidino- and Related Amino Acids succinate, all containing no a-amino group, Enzyme hardly affected the reaction at 1 mM.Citrulline, Addition activity Inhibition homocitrulline and ornithine strongly inhibit- ( 1 niM) Gumol/mg (%) protein/min) ed the enzymereaction while proline, glutamate and urea had slightly inhibitory effects. None 1.10 0 Guanidine HC1 1.03 6.8 Guanidinoacetate 1.05 5.0 DISCUSSION /?-Guanidinopropionate 1. 1 0 0 y-Guanidinobutyrate 0. 88 20. 1 Arginine deiminase in E. gracilis was found Guanidinosuccinate 1. 1 3 - 3.0 to be located in mitochondria from its parallel L-a-Amino-^-guanidino- Q ^ 74 distribution with succinate semialdehyde dehy- propionate D-Arginine 0.42 61.5 drogenase, a marker enzymefor the mitochon- L-Homoarginine 0.48 56.8 drial matrix. Parallel behavior of the two Agmatine 0.98 1 1. 1 enzymes in sonication of isolated Euglena mi- Arginosuccinate 0. 96 1 2. 7 tochondria supports the conclusion. Hill and Citrulline 0.5 1 53.5 Homocitrulline 0. 64 42. 1 Chambers8} have reported that the deiminase Ornithine 0. 33 70.4 in Tetrahymena is located in the 100,000xg Apartate 1.12 å -2.0 supernatant of freeze-thawed cells. Glutamate 0. 87 20. 6 Arginine deiminase of E. gracilis was pu- Proline 0.74 32.4 rified about 56-fold. This is the first case of Fumarate 1. 14 -4.0 Urea 0.87 21.2 purification of the deiminase to homogeneity from an eukaryote. Properties of this enzyme have been studied mainly in bacteria, such as from this inhibition by 2-mercaptoethanol, Mycoplasma^ and Pseudomonas.6) The molec- indicating that a sulfhydryl group in the en- ular weight of the purified Euglena enzyme zyme protein is concerned with the active was estimated to be 87,000 by gel filtration, center of the enzyme. Isonicotinic acid hy- and the result of acrylamide gel electrophoresis drazide, aminoxyacetic acid, and semicarba- in the presence of SDS suggested that the zide, the inhibitors of vitamin B6 enzymes, enzymeconsisted of two apparently identical hardly affected the deiminase activity (Table subunits with a molecular weight of 48,000. IV). The Euglena enzyme was distinct from the The enzyme of Mycoplasma hominis4) has a Streptococcus enzyme5) in that it was not molecular weight of 78,300 and that of inhibited by carbonyl reagents. Pseudomonas putida6) 120,000. The Psedo- 488 B.-S. Park et al.

monasenzymewas shownto be consisted of parently regulated by a unique mechanism two identical subunits.6) depending on the characteristic property of Euglena deiminase showed its highest ac- arginine deiminase. It has been found that tivity at pH 9.7 to 10.3, distinct from the phosphoenolpyruvate carboxylase in enzymes from yeast,7) bacteria,6) Chlorella18) Escherichia coli20) and rabbit muscle and Mycoplasma^which have optimum pHs glyceraldehyde-3-phosphate dehydrogenase21 ) at 6.0 to 6.7. The Tetrahymena enzyme8) is exhibit negative cooperativity in the binding most active at pH 9.3 and Chlamydomonas with substrate. Effects of L-arginine analogues enzyme9) at 8.0~8.6. the Euglena deiminase containing guanidino groups are classified into required Co2+for the reaction and the Km the two following froups: (a) L-a-amino-/?- value for Co2+ was 0.23mM. The metal ion guanidinopropionate, D-arginine, and l- requirement of arginine deiminase is reported homoarginine, which are a-amino acids and here for the first time. Roche and Lacombe7) strongly inhibit arginine deiminase activity reported that arginine deiminase obtained by and (b) jS-guanidinopropionate, y-guanidino- plasmolysis of baker's yeast was inactivated by butyrate, and guanidinosuccinate which con- dialysis and reactivated by the addition of tain no a-amino group and hardly inhibit the Co2+ or Ni2+, but that such an effect of metal enzyme. The results suggest that an a-amino ions was not found in the alcohol-fractionated group is essential for the binding of arginine or enzymefrom acetone-treated yeast. They con- an analogue to the catalytic site of the enzyme. cluded that the effect was due to stabilization Shibatani et al6) have reported that the ar- of the active structure of the enzyme by the ginine deiminase from P. putida is not in- metal ion. hibited by D-arginine. Inhibition of Euglena Euglena arginine deiminase was markedly arginine deiminase by citrulline and ornithine inhibited by sulfhydryl reagents, and the in- indicates that these compounds serve as reg- hibition was removed by 2-mercaptoethanol, ulatory end products in the arginine dihy- indicating that a sulfhydryl group is involved in the active site of the enzyme. The drolase pathway. Pseudomonasenzyme6)has also been reported REFERENCES to be inhibited by sulfhydryl inhibitors. 1) B.-S. Park, A. Hirotani, Y. Nakano and S. Kitaoka, Euglena deiminase was revealed to be Agric. Biol. Chem., 47, 2561 (1983). unique in that negative cooperativity was ob- 2) C. H. vanEtten,W. F. Kwolek,J. E. PetersandA. S. served, indicating that substrate-induced con- Barclay, J. Agric. Food Chem., 15, 1077 (1967). formational changes are responsible for the 3) R. L. Weiss, /. BacterioL, 126, 1173 (1976). 4) J. L. Weickmann and D. E. Fahrney, J. Biol. Chem., regulation of the activity of this enzyme.19) In 252, 2615 (1977). a typical experiment, when Euglena cells reach- 5) B. Petrack, L. Sullivan and S. Ratner, Arch. Biochem. ed the early exponential phase of growth the Biophys., 69, 186 (1957). intracellular concentration of arginine was 6) T. Shibatani, T. Kakimoto and I. Chibata, /. Biol. 190mM,1*and the amino acid decreased mark- Chem., 250, 4580 (1975). edly in the mid-exponential phase; in the late 7) J. Roche and G. Lacombe, Biochim. Biophys. Acta, 9, exponential phase the arginine level increased 687 (1952). 8) D. L. Hill and P. Chambers, Biochim. Biophys. Acta, again rapidly to reach about 30mM. When 148, 435 (1967). ethanol was added as a carbon source in the 9) J. S. Sussenbach and P. J. Strijkert, FEBS Lett., 3, late exponential phase, the size of the arginine 166 (1969). pool was rapidly decreased and renewed cell 10) M. Tokunaga, Y. Nakano and S. Kitaoka, J. Protozool, 26, 471 (1979). division took place. These facts indicate that ll) S. Shigeoka, Y. Nakano and S. Kitaoka, Agric. Biol. arginine is accumulated as a nitrogen reserve Chem., 43, 2187 (1979). in Euglena, and its rapid accumulation and 12) M. M. Bradford, Anal. Biochem., 72, 248 (1976). mobilization along with cell growth is ap- 13) M. Tokunaga, Y. Nakano and S. Kitaoka, Biochim. Arginine deiminase in Euglena gracilis 489

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