ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 290, No. 1, October, pp. 46-50, 1991

The C-S Lyases of Higher Plants: Homogeneous ,&Cystathionase of Spinach Leaves’ August L. Staton and Mendel Mazelis’ Department

of Food Science and Technology, University

of California, Davis, California

95616

Received March 20, 1991, and in revised form May 28, 1991

S-Substituted cysteines and their derivatives are prominent secondary amino acids in a number of plant families. The substituents are often specific and unique to each family. Cystathionine, however, is an ubiquitous S-substituted cysteine found in all autotrophic plants since it is an intermediate in the biosynthesis of methionine. /?-Cystathionase will produce homocysteine and pyruvate from cystathionine by a &elimination reaction. The present report describes the purification of this enzyme to homogeneity from spinach leaves and some of its properties. The enzyme has a molecular weight of 210,000 and consists of four identical subunits of M, 53,000. It has a pH optimum for activity of 8.6-8.7 and utilizes pyridoxal-5’-phosphate as a cofactor. Its specificity is limited to L-cystathionine, L-djenkolate, and Lcystine as substrates with a relative activity of 100: 126: 17, respectively. It is not a glycoprotein unlike a number of previously described plant C-S lyases. o 1991 Academic Press,

Inc.

Secondary nonprotein sulfur-containing amino acids are common constituents in many higher plants (1). The characteristic flavors and odors of a number of important vegetables are due to the enzymatic degradation of these amino acids such as alkyl cysteines and their sulfoxides (2). The enzymes cleave the C-S bond to produce a sulfurcontaining product, pyruvate, and ammonia. Well known examples of such enzymes have been purified and characterized from garlic (3), onion (4), and leek (5) of the monocot genus Allium. Analogous enzymes have also been purified from cabbage (6) and broccoli (7) of the dicot genus Brassica and from the legumes Albizzia lophanta (8) and Acacia farnesiana (9). The enzymes show different relative substrate specificities, but all are capable of 1 This paper is part of a series “The C-S Lyases of Higher Plants.” The preceding paper in this series is Ref. (5). ’ To whom correspondence should be addressed.

46

cleaving S-alkyl cysteine sulfoxides. Since these enzymes are found in taxonomically unrelated families in both the monocots and the dicots, the degree of homology in these enzymes was of interest. The enzymes from three species of Allium, viz. garlic (A. satiuum), onion (A. cepa), and leek (A. porrum), were found to differ significantly in molecular weight. Garlic and onion enzymes had much different pH optima for activity. Polyclonal antibodies to the garlic enzyme would not interact with the enzyme from onion or leek (10). Similar results were obtained with onion enzyme antibodies and the garlic and leek enzymes. There is one S-substituted cysteine which is ubiquitous in autotrophic higher plants. Cystathionine is a key intermediate in methionine biosynthesis (11). The enzyme P-cystathionase which cleaves cystathionine to homocysteine, pyruvate, and ammonia should be present in all higher plants. The presence of this enzyme in crude extracts from a number of higher plants has been demonstrated and its partial purification from spinach (Spinucia oleracea) leaves reported (12). In order to study the degree of homology of this ubiquitous enzyme from various species, it is necessary to purify the enzyme from one source to homogeneity for comparative purposes. Based on the earlier work of Giovanelli and Mudd (12), spinach leaves would be a convenient source of active enzyme. The present report describes the purification of homogeneous pcystathionase from spinach leaves and its characterization. MATERIALS

AND

METHODS

Chemicals. L-Cystathionine, L-djenkolate (L-5,5’-methylenebis [cysteine]), PLP,3 PVPP, octyl-Sepharose CL-4B, QAE-Sephadex, andprotein gel filtration standards were obtained from Sigma Chemical Co. L-

3 Abbreviations used: buffer A, 0.1 M potassium phosphate-O.01 mM PLP (pH 7.2); PAGE, polyacrylamide gel electrophoresis; PLP, pyridoxal-5’-phosphate; PVPP, polyvinylpolypyrollidone; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Copyright Q 1991 AI1 rights of reproduction

0003.9861/91 $3.00 by Academic Press, Inc. in any form reserved.

HOMOGENEOUS

@-CYSTATHIONASE TABLE

Purification

Procedure Homogenate Ammonium sulfate Octyl-Sepharose QAE-Sephadex

I

Table of fl-Cystathionase

from Spinach

Total units

Protein h4

Specific activity

877

2193

53 44 4

2126 1539 282

5560 363 23 0.23

0.39 5.86 66.04 1226.1

Volume (ml)

Note. One unit of enzyme activity

produces 1 nmol of pyruvate

per minute at 22°C. Specific activity

and meso-lanthionine, L-cysteine, DL-homocysteine, DL-homocystine, and Aquacide II were purchased from Calbiochem. L-Cystine was a product of Nutritional Biochemical Corp., ultrapure ammonium sulfate was purchased from Schwarz-Mann Biotech., SDS-PAGE molecular weight standards were obtained from Bio-Rad, and 2,4-dinitrophenylhydrazine was a product from Eastman Kodak. The alkyl-cysteine sulfoxide substrates were synthesized from the thioethers as previously described (13). Pur$ication protocol. Spinach leaves were purchased from local markets as needed. All operations were carried out in the cold. Spinach leaves were washed with deionized water and the petioles removed. The leaves were blended for 30 s in buffer A containing 0.1 mM EDTA, and 5% PVPP (w/v) using 1.5 ml of buffer for each gram of leaf. 1. The homogenate was strained through two layers of cheesecloth and centrifuged at 10,OOOgfor 30 min at 4°C. The supernatant solution was brought to 25% saturation with (NH&SO, and stirred for 15 min. The solution was then centrifuged at 13,000g for 20 min and the supernatant solution decanted. This was now brought to 35% saturation with (NH&SO,. The resulting precipitate was sedimented by centrifugation as above and suspended in a minimal amount of buffer A. Any undissolved material was removed by centrifugation. 2. A column (3 X 14 cm) of octyl-Sepharose Cl-4B was prepared and equilibrated with a solution of buffer A containing 15% (NH&SO, at a flow rate of 8 ml/min. The soluble fraction from step 1 above was made 10% saturated with ammonium sulfate and applied to the column. The column was washed with 4 bed vol of buffer A containing 11% (NH&SO, and the eluant was discarded. The enzyme was eluted with a solution of buffer A-5% (NH&Sod. 3. The active fractions from the column were pooled together and concentrated by ultrafiltration to 2 ml. Deionized water was added to make the volume to 30 ml and the solution concentrated again. This served to desalt the sample. A column (1.2 X 10 cm) of QAE-Sephadex was prepared by the guidelines furnished by the manufacturer (14). The column was equilibrated with 0.005 M potassium phosphate buffer (pH 7.8) containing 0.01 mM PLP. The desalted sample was then placed on the column and the column washed with 4 bed vol of the equilibrating buffer containing 0.2 M NaCl at a flow rate of 0.07 ml/min. A linear gradient from 0.2 to 0.3 M NaCl was then applied with this buffer. The active fractions were pooled and concentrated to 2 ml by dialysis against Aquacide II. This fraction was used for further characterization. Enzyme assay. The usual assay reaction mixture consisted of 2.5 pmol substrate, 20 Fmol Tris buffer, pH 8.6, and enzyme in a final volume of 0.25 ml. The mixture was incubated at room temperature (23°C) for 30 min and terminated by the addition of 0.5 ml 10% (w/v) trichloroacetic acid. It had been determined that the reaction was linear under these conditions for 60 min. The precipitated protein was removed by centrifugation. The supernatant solution was assayed for the product, pyruvate, calorimetrically (15). Giovanelli and Mudd (12) had shown that the spinach leaf cystathionase utilized L-djenkolate as readily as L-cystathionine. Because of convenience and cost L-djenkolate was used as

47

OF SPINACH

Purification

Recovery %

15

97 70 13

169 3144

is in units per milligram

of protein.

the substrate for the assay during the purification and activity with Lcystathionine checked at each step. Protein determinations. Q uan t’ti a t’iv e protein measurements were made by the method of Bradford (16) using bovine serum albumin as a standard. Ekctrophretic analysis. Polyacrylamide gel electrophoresis (PAGE) was used to analyze protein samples during the purification procedure. Both dissociating and nondissociating gels were prepared and run as described by Hames (17). In nondissociating PAGE the protein samples were in 10% glycerol and 0.002% bromphenol blue was added as a tracking dye. SDS-PAGE was used for subunit analysis. The protein samples were denatured and prepared for electrophoresis by heating at 100°C for 4 min in the sample buffer consisting of 50 mM Tris-HCl, pH 6.8, 2% SDS, 1% mercaptoethanol, 0.001% bromphenol blue, and 10% glycerol. The gels were stained by Coomassie blue using the one-step procedure of Zehr et al. (18). They could subsequently be stained by AgNO, (19) for increased sensitivity. Densitometer tracings of the stained gels were obtained by use of an LKB Ultrascan XL laser densitometer.

0.303 4A

B i

0.081

Top of Gel

FIG. 1. Densitometer tracing of purified enzyme run by nondissociating PAGE stained with (a) Coomassie blue or (b) silver stain.

48

STATON

AND

MAZELIS

0.156 4.6

4.2

a 1.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Rf FIG. 4. Subunit molecular weight estimation by SDS-PAGE. The following proteins were used: A, bovine serum albumin; B, ovalbumin; C, carbonic anhydrase (31,000); D, soybean trypsin inhibitor (21,500); E, lysozyme (14,400).

RESULTS Topof

Gel

FIG. 2. Densitometer tracings of purified enzyme run by SDS-PAGE stained with (a) Coomassie blue or (b) silver stain.

The molecular weight of the native Molecular weight determimtiom. holoenzyme was estimated using a standardized gel filtration column (20). Proteins used as standards and their (molecular weights) included myoglobin (M, 17,000). ovalbumin (44,000), bovine serum albumin (65,400), y-globulin (158,000), and thyroglobulin (670,000). Subunit molecular weights were determined by SDS-PAGE and known molecular weight standards. The protein standards used were lysozyme (14,400), soybean trypsin inhibitor (21,500), carbonic anhydrase (31,000), ovalbumin, bovine serum albumin, and phosphorylase B (92,500).

A typical purification from 700 g of spinach leaves is summarized in Table I. The final fraction was purified over 3000-fold with a 13% yield. The enzyme could be stored at -10°C for 1 month without any appreciable loss of activity. Repetitious freezing and thawing did reduce the activity. To alleviate this problem the final fraction was divided into small portions which were then frozen and used individually. Stability could also be increased

TABLE Amino

II

Acid Composition

nmol/50 ~1

pmol/mg protein

1.843 0.964 3.594

0.670 0.350

mol of amino acid/m01 protein

6R

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cysteine

FIG. 3. Molecular weight estimation by gel filtration. The protein standards used were A, thyroglobulin (670,000); B, y-globulin (158,000); C, bovine serum albumin (65,000); D, ovalbumin (44,000); E, myoglobin (17,000).

Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan

0.969 5.296 2.579

0.352

140.75 73.58 274.48 416.80 74.00

1.926 0.938

404.39 196.93

0.842

0.306

64.32

0.190 0.461 0.859

0.069

14.51

0.168

0.378 0.358 0.404 0.244

0.137 0.130 0.147 0.089

0.451 b

0.164

35.22 65.60 28.87 27.32 30.82 18.60 34.42

5.458

0

1.307 1.985

0.312

’ Not detected: less than 0.15 nm/50 microliters b Not determined.

if present.

HOMOGENEOUS TABLE

P-CYSTATHIONASE

to 9.2. The optimum activity was found at pH 8.6-8.7. All assays were run at this pH.

III

Substrate Specificity Relative

Substrate L-Cystatbionine L-Djenkolate L-Cysteine S-Methyl-L-cysteine S-Ethyl-L-cysteine L-Cystine L-Homocystine L-Lanthionine

sulfoxide sulfoxide

Note. Each substrate was assayed at a concentration

activity 100 126 0 0 0 17 0 0 of 10 mM.

by concentrating the enzyme solution by ultrafiltration or the addition of small amounts of bovine serum albumin. Determination

of Homogeneity

The holoenzyme was analyzed by PAGE and staining by Coomassie blue and also with silver stain. The gels were then scanned by a densitometer. The results of the scans are shown in Fig. 1. Both stains revealed only one major band. The enzyme was also analyzed by SDSPAGE and visualized by Coomassie blue and silver staining. The densitometer scans of these gels (Fig. 2) showed only one major peak. Molecular

49

OF SPINACH

Weight Determinations

The molecular weight of the cystathionase was determined by using a gel filtration column standardized by measuring the elution volumes of proteins of known molecular weight. The holoenzyme had a molecular weight of 210,000 (Fig. 3). The use of SDS-PAGE and known molecular weight markers with this technique showed there was one subunit of n/i 53,000 (Fig. 4). This result strongly suggests that the holoenzyme is composed of a tetramer of equal subunits.

Substrate Specificity

and K,,, Values

Several S-substituted cysteines found commonly in a number of higher plants were tested as potential substrates for the cystathionase. Presumed saturating amounts of substrate (0.01 M) were used in each assay. The relative activities of each substrate to cystathionine are presented in Table III. Only L-cystathionine, L-djenkolate, and L-cystine of the substrates tested had activity. The apparent K,,, values for L-cystathionine and Ldjenkolate were determined by varying the concentration of each substrate within a range of 0.1 to 3.0 mM. The K,,, values were then determined by a linear regression evaluation of the straight line relationship of the reaction. The K, for I,-cystathionine was 0.30 and 0.35 mM for Ldjenkolate. The relative V,,, of djenkolate was 129% of that of cystathionine, which is in agreement with the value in Table III and that reported by Giovanelli and Mudd (12). Activation

Energies

The activation energy for each of the three substrates was obtained by the use of an Arrhenius plot. It was found that the activity of the enzyme increased in a linear fashion with increasing temperature to 50°C. Each substrate was then run with the enzyme at a series of temperatures between 22 and 45°C for 15 min. The activation energies were then calculated from the relationships shown in Fig. 5. The activation energies were almost identical for each substrate being 64 kJ/mol for cystathionine, and 66 and 69 kJ/mol for cystine and djenkolate, respectively. DISCUSSION

Several enzymes capable of carrying out the P-cleavage of S-substituted cysteines have been previously identified

Amino Acid Composition The amino acid composition of cystathionase is summarized in Table II. Since the amino acid analysis was carried out on an acid-hydrolyzed sample, tryptophan could not be determined. Cysteine and methionine are also recovered in low amounts by this particular procedure. The other amino acids are given as micromoles/ milligram protein and in moles/mole of enzyme based on a molecular weight of 210,000. pH Optimum The pH optimum for the activity of the purified enzyme was determined with Tris buffer from a pH range of 7.8

-7 0.0031

0.0032

0.0033

0.0034

l/T FIG. 5. Determination of the energy of activation with different substrates. k is the rate of pyruvate formation in moles per second. (0) Cystathionine; (w) djenkolate; (0) cystine.

50

STATON

AND

and characterized from higher plants. In all such cases cystathionine, which is an ubiquitous S-substituted cysteine, has been a very poor substrate. In the present work we have purified an enzyme from spinach leaves to apparent homogeneity. This enzyme has high activity with cystathionine and djenkolate and very limited specificity for other cysteine derivatives. This distinguishes it from the cystine lyases of the Cruciferae (6, 7), alliin lyases of Allium sp. (3-5), and the C-S lyases of A. lophanta (8) and A. farnesiana (9). Cystathionases have previously been partially purified and characterized from pig liver (21), Proteus morganii (al), Salmonella typhimurium (22), Escherichia coli (23), Neurospora (24), and spinach leaves (12). Previous studies had shown the spinach enzyme to be quite unstable after several purification steps (12). We found that concentration of the enzyme by ultrafiltration or the addition of an inert protein such as bovine serum albumin increased the stability of the enzyme to storage. Inclusion of pyridoxal phosphate in all the buffers used in the isolation also led to a greater retention of activity. Storage at -10°C maintained activity for up to 1 month. However, frequent thawing and refreezing caused a rapid loss of activity. The purified spinach enzyme showed a more limited substrate specificity than the bacterial and Neurospora enzymes. Only L-djenkolate and L-cystine were cleaved to any degree in addition to cystathionine. D-Cystathionine was inactive as a substrate. The pH optima and K,,, values for L-cystathionine were quite similar to previously described cystathionases. Since cystathionine is found as an intermediate in methionine biosynthesis the /3-lyase should be present in all autotrophs. The other plant CS lyases described above showed no cross-reaction immunologically to garlic alliin lyase or onion alliin lyase polyclonal antibodies (10). The preparation of a homogeneous P-cystathionase will allow further studies on the comparative homology of this enzyme from different species and with other C-S lyases. REFERENCES 1. Rosenthal, G. A. (1982) Plant Nonprotein Academic Press, New York.

Amino and Imino Acids,

MAZELIS 2. Schwimmer, S. (1981) Source Book of Food Enzymology, lishing Co., Westport.

Avi Pub-

3. Neck, L. P., and Mazelis, M. (1986) Arch. Biochem. Biophys. 249, 27-33. 4. Neck, L. P., and Mazelis, M. (1987) Plant Physio2. 85,1079-1083. 5. Won, T., and Mazelis, M. (1989) Physiol. Plant 77, 87-92. 6. Hall, D. I., and Smith, I. K. (1983) Plant Physiol. 72, 654-658. 7. Hamamoto,

A., and Mazelis, M. (1986) Plant Physiol. 80,702-706.

8. Schwimmer, 316-324.

S., and Kjaer, A. (1960) Biochim. Biophys. Acta 42,

9. Mazelis, M., and Creveling, R. K. (1975) Biochem. J. 147,485-491. 10. Neck, L. P., and Mazelis, M. (1989) Phytochemktry

28, 729-731.

11 Giovanelli, J. G., Mudd, S. H., and Datko, A. (1980) in The Biochemistry of Plants (Stumpf, P. K., and Conn, E. E., Eds), Vol. 5, pp. 453-505, Academic Press, San Diego. 12. Giovanelli, J. G., and Mudd, S. H. (1971) Biochim. Biophys. Acta 227, 654-670. 13 Stoll, A., and Seebeck, E. (1951) Adv. Enzymol. 14. Pharmacia Fine Chemicals. QAE-Sephadex, exchanger.

11, 377-400.

a fully quaternized

ion

15. Mazelis, M., Beimer, N., and Creveling, R. K. (1967) Arch. Biochem. Biophys. 120,371-378. 16. Bradford,

M. M. (1976) Anal. Biochem. 72, 248-254.

17. Hames, B. D. (1981) in Gel Electrophoresis of Proteins-A Practical Approach (Hames, B. D., and Rickwood, D., Eds.), IRL Press, London/Washington. 18. Zehr, B. D., Savin, T. J., and Hall, R. E. (1989) Anal. Biochem. 182, 157-159. 19. Heukeshoven, 112. 20. Whitaker,

J., and Dernick,

R. (1985) Electrophoresis

6, 103-

J. R. (1963) Anal. Chem. 35, 1950-1953.

21. Binkley, F. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., Eds.), Vol. 2, p. 314, Academic Press, New York. 22. Guggenheim, S. (1971) in Methods in Enzymology (Tabor, H., and Tabor, C. W., Eds.), Vol. 17B, pp. 439-442. Academic Press, New York. 23. Uren, J. R. (1987) in Methods in Enzymology (Jakoby, W. B., and Griffith, 0. W., Eds.), Vol. 143, pp. 483-486, Academic Press, San Diego. 24. Flavin, M. (1971) in Methods in Enzymology (Tabor, H., and Tabor, C. W., Eds.), Vol. 17B, pp. 450-453, Academic Press, New York.