Vol. 163, No. 2JOURNAL OF BACTERIOLOGY, Aug. 1985, p. 669-6760021-9193/85/080669-08$02.00/0Copyright C 1985, American Society for Microbiology

Purification and Characterization of Selenocysteine 3-Lyase fromCitrobacter freundii

PATRICK CHOCAT,t NOBUYOSHI ESAKI, KATSUYUKI TANIZAWA, KAORU NAKAMURA, HIDEHIKOTANAKA, AND KENJI SODA*

Institute for Chemical Research, Kyoto University, Uji, Kyoto-Fu 611, Japan

Received 26 September 1984/Accepted 7 May 1985

The purification and characterization of bacterial selenocysteine (-lyase, an enzyme which specificallycatalyzes the cleavage of L-selenocysteine to L-alanine and Se, are presented. The enzyme, purified to nearhomogeneity from Citrobacterfreundii, is monomeric with a molecular weight of ca. 64,000 and contains 1 molof pyridoxal 5'-phosphate as a cofactor per mol of enzyme. L-Selenocysteine is the sole substrate (K,, 0.95 mM).L-Cysteine is a competitive inhibitor of the enzyme (Ki, 0.65 mM). The enzyme also catalyzes the a,"elimination of 0-chloro-L-alanine to form NH3, pyruvate, and Cl- and is irreversibly inactivated during thereaction. The physicochemical properties, e.g., amino acid composition and subunit structure, of the bacterialenzyme are fairly different from those of the pig liver enzyme (Esaki et al., J. Biol. Chem. 257:43864391,1982). However, the catalytic properties of both enzymes, e.g., substrate specificity and inactivation by thesubstrate or a mechanism-based inactivator, P-chloro-L-alanine, are very similar.

Selenium is an essential micronutrient for mammals,birds, and some bacteria, and a selenocysteine (2-amino-3-hydroselenopropionic acid) or selenomethionine [2-amino-4-(methylseleno)-butyric acid] residue has been found in thepolypeptide chain of several bacterial and mammalian sele-nium-dependent enzymes (7, 14, 18, 21). However, littleattention has been paid to the biosynthesis and metabolismof selenium amino acids.During the course of a study of selenocysteine synthesis in

rat livers (13), we found a novel pyridoxal 5'-phosphate(pyridoxal-P) enzyme that catalyzes specifically the P elim-ination of L-selenocysteine into L-alanine and elementalselenium and named it selenocysteine P-lyase (12). Theenzyme occurs widely in various mammalian tissues (12) andin the cells of aerobic bacteria (6); the pig liver enzyme hasbeen purified to homogeneity (12).To understand the properties of the bacterial enzyme, we

have purified it to near homogeneity from the cell extract ofCitrobacterfreundii. We describe here the physicochemicaland enzymological characteristics of the bacterialselenocysteine P-lyase and compare them with those of thepig liver enzyme presented previously (12).

MATERIALS AND METHODSMaterials. DL-Selenocystine, P-chloro-L-alanine, O-acetyl-

L-serine, L-serine-O-sulfate, phenylmethylsulfonyl fluoride,selenocystamine, RNase, and DNase were purchased fromSigma Chemical Co., St. Louis, Mo., and dithiothreitol andpyridoxal-P were from Nakarai Chemicals, Kyoto, Japan.L-Selenocystine and DL-[a-2H]selenocystine were synthe-sized as described previously (N. Esaki, N. Karai, T.Nakamura, H. Tanaka, and K. Soda, Arch. Biochem.Biophys., in press), as were DL-selenohomocysteine andL-selenocystathionine (13). Selenium ethyl-L-selenocysteinewas prepared from L-selenocysteine with ethyl iodide (13).L-Selenocystine ethyl ester, L-propargylglycine, andhydroxyapatite were prepared by the methods of Biemann et

* Corresponding author.t Present address: ELF-Bio Recherches, Labege, 31320 Casta-

net-Tolosan, France.

al. (2), Abeles and Walsh (1), and Tiselius et al. (33),respectively. Selenols were prepared from the correspondingdiselenides by reduction in situ with a fivefold excess ofdithiothreitol (17). The other chemicals were analytical-gradereagents.

Alanine dehydrogenase (EC 1.4.1.1) was purified to ho-mogeneity from a cell extract of Bacillus stearothermophilus(IFO 12550) (Y. Sakamoto, S. Nagata, K. Inagaki, T.Oshima, H. Tanaka, and K. Soda, Abstr. Annu. Meet.Agric. Chem. Soc. Jpn. 1984, 1H-26, p. 113). Amino acidracemase with low substrate specificity (EC 5.1.1.10) waspurified to near homogeneity as described in the literature(19).

Preparation of DL-[a-3H]selenocystine. DL-[a-3H]-selenocystine was prepared by racemization of DL-selenocystine in tritiated water with amino acid racemasethat has low substrate specificity. The amino acid (10 ,umol)was incubated with 0.5 ,ug of the enzyme at 37°C for 12 h ina reaction mixture containing 0.1 mmol of potassium phos-phate buffer (pH 7.2), 10 nmol of pyridoxal-P, and 0.1 ,umolof EDTA in a final volume of 1 ml of tritiated water (9 ,uCi).As the enzyme is irreversibly inactivated by L-selenocys-teine (unpublished data), no reductant was added in thereaction mixture. However, as selenocystine is easily oxi-dized at neutral or alkaline pH, oxygen was purged from thereaction mixture by thorough flushing with N2 before theaddition of the enzyme. The reaction was stopped by theaddition of 20 jil of 6 N HCl; after centrifugation at 10,000 xg for 5 min, the sample was charged on a Dowex 50X8column (H' form) (10 by 30 mm). After being washedsuccessively with 40 ml each of water, 2 N HCI, and water,the amino acid was eluted with 20 ml of 2 N NH40H. Thesample was dried under reduced pressure and dissolved into1 ml of 1 N HCl twice to remove exchangeable 3H. Thesample was kept at -20°C until use. DL-[a-3H]seleno-cystine (1.8 ,umol; specific activity, 1.1 x 106 dpm/4Lmol) wasthus prepared. The racemase catalyzes only the exchange ofthe a-proton and not the a-protons of the substrate with 2Hin 2H20 when analyzed by 'H-nuclear magnetic resonance(unpublished data). Therefore, tritium is most probablyincorporated only into the a position of selenocystine.

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Enzyme assays. Selenocysteine 3-lyase was assayedspectrophotometrically by measuring the amount of alanineformed with alanine dehydrogenase in the presence of NAD(29). The standard reaction mixture contained 0.5 ,umol of L-

or DL-selenocystine, 2.5 pmol of dithiothreitol, 10 ,g ofbovine serum albumin, 25 ,umol of phosphate buffer (pH7.2), 50 nmol of pyridoxal-P, and enzyme in a final volume of0.25 ml. Alternatively, the enzyme activity was measured bythe determination of H2Se with lead acetate as describedpreviously (12). In this case, the phosphate buffer was

replaced by 0.1 M sodium hydroxide-borate buffer (pH 7.2)in the reaction mixture, as phosphate and lead ions form a

precipitate under the assay conditions.For the identification of the selenium compound evolved

from selenocysteine, the enzyme reaction was carried outwith a reaction mixture that did not contain dithiothreitol.This mixture was prepared by reduction of 20 ,umol ofL-selenocystine with a fivefold molar excess of NaBH4 in 8ml of distilled water at room temperature for 15 min. Theexcess NaBH4 was then decomposed by lowering the pH toca. 2 with 6 N HCI, and the mixture was kept at room

temperature for 15 min. The pH was then adjusted to ca. 7.0with 10 N NaOH, and 1 mmol of sodium borate buffer (pH7.2), 0.5 ,mol of pyridoxal-P, and 1 mg of bovine serum

albumin were added to the solution. All of these operationswere carried out under a constant stream of 02-free N2, andthe mixture was used immediately after preparation. Ele-mental selenium was determined with acidic lead acetateafter reduction with excess NaBH4 (12).

Kynureninase (EC 3.7.1.3), aspartate 3-decarboxylase(EC 4.1.1.12), cystathionine ,B-synthase (EC 4.2.1.22), cys-

tathionine y-lyase (EC 4.4.1.1), alanine aminotransferase(EC 2.6.1.2), and O-acetylserine (thiol)-lyase (EC 4.2.99.8)were assayed as described previously (12). Amino acidswere determined by amino acid analysis (32) except cyste-ine, which was determined by the method of Gaitonde (15),and a-keto acids were determined by the method of Soda(31). The activity of the enzyme on selenocysteine analogswas examined by carrying out the reaction in a standardreaction mixture in which selenocysteine was replaced bythe analog. For amino acid substrates, the formation of theproduct amino acids or the disappearance of the substratewas followed. For the other substrates, such as cysteamine,selenocysteamine, and L-selenocysteine ethyl ester, the for-mation of H2S or H2Se was monitored with lead acetate (3,12, 28).One unit of selenocysteine P-lyase is defined as the

amount of enzyme that catalyzes the appearance of 1 Fmolof H2Se or alanine per min. Specific activity is expressed as

units per milligram of protein. Protein was determined by themethod of Lowry et al. (26), with bovine serum albumin as a

standard.Inactivation by j1-chloro-L-alanine. Selenocysteine 3-lyase

(9.6 pRg) was added to a solution containing an appropriateconcentration of P-chloro-L-alanine (L-Cl-alanine) in 100 mMpotassium phosphate buffer (pH 7.2) at 37°C. Samples (5 RIl)were removed at intervals and diluted 50 times with thestandard reaction mixture described above to determine theremaining activity.

Reaction in D20. The amino acid (2 ,Lmol) was incubated at37°C for 2 h with 0.1 U of selenocysteine ,-lyase in a

reaction mixture (1 ml) containing 0.1 mmol of potassiumphosphate buffer (pD 7.2), 10 ,umol of dithiothreitol, 10 ,umolof pyridoxal-P, and 0.1 ,umol of EDTA in D20. The enzymewas dialyzed for 4 h against 1,000 volumes of the reactionmixture in which the substrate was omitted before being

added to the reaction mixture. The reaction was stopped bythe addition of 20 ,ul of 6 N HCl, and the denatured proteinwas removed by centrifugation at 10,000 x g for 5 min.When L-selenocysteine was used as the substrate, the re-maining selenocysteine and alanine were separated by chro-matography with a Dowex 50X8 (H+ form) column (10 by 20mm). All samples were analyzed by 1H-nuclear magneticresonance and by gas chromatography-mass spectrometry(GC-MS). To determine the 2H distribution in alanine, thesample was analyzed before and after racemization in wateras follows. The amino acid was incubated with 0.5 ,g ofamino acid racemase with low substrate specificity in 1 ml of0.1 M potassium phosphate buffer (pH 7.2) at 37°C for 12 hand was isolated with the same Dowex 50X8 column asdescribed above.Reaction with DL-[U_3lHselenocystine. DL-[a-3H]-

selenocystine (1.8 ,mol) was incubated at 37°C for 2 h with0.16 U of selenocysteine P-lyase in D20 as described above.The reaction was stopped by the addition of 20 RI of 6 NHCl, and the denatured protein was removed by centrifuga-tion at 10,000 x g for 5 min. The mixture was charged on aDowex 50X8 (H+ form) column (10 by 20 mm). Afterwashing with 40 ml of water, alanine was eluted with 20 mlof 2 N HCl. The sample was dried under reduced pressureand dissolved in 1 ml of water. This was repeated twice toremove all exchangeable tritium. The isolated alanine wasfurther analyzed by thin-layer chromatography with a sol-vent system (ethanol-water, 1:1 [vol/vol]) which allows clearseparation between alanine and selenocystine (Rf = 0.33 and0.08, respectively). To determine the position of tritium inlabeled alanine, the isolated amino acid was racemized inwater and isolated as described above; the remaining radio-activity was then determined.

Analytical methods. Disc gel electrophoresis was per-formed in 7.5% polyacrylamide gel by a modification of theprocedure of Davis (9). The molecular weight (MW) wasestimated by high-pressure liquid chromatography with aTSK gel G3000 SW column (7.53 by 600 mm) (Toyo SodaManufacturing Company, Tokyo, Japan). For calibration,glutamate dehydrogenase (MW, 290,000), lactate dehydro-genase (MW, 142,000), enolase (MW, 70,000), adenylatekynase (MW, 32,000), and cytochrome c (MW, 12,400) fromOriental Yeast Co., Osaka, Japan, were chromatographedunder the same conditions. The MW of the subunit wasestimated by disc gel electrophoresis in the presence of0.1%sodium lauryl sulfate (25). The isoelectric point of theenzyme was determined by isoelectric focusing on a 5%polyacrylamide gel column containing 2% Ampholine (LKBInstruments Inc., Rockville, Md.; pH range, 3.5 to 8) (35).The pH gradient was determined by soaking 2-mm slices ofa blank gel in distilled water.For amino acid composition analyses, the Citrobacter

freundii enzyme (4.2 ,g) and the pig liver enzyme (24 p,g)were dialyzed against 1,000 volumes of 5 mM potassiumphosphate buffer (pH 7.2) before lyophilization and thenhydrolyzed in 6 N HCI at 110°C for 24 h in evacuated sealedtubes. The analyses were performed on a Hitachi high-performance amino acid analyzer (model no. 835) with astandard program (30).The optical purity of amino acids was analyzed by reverse-

phase chromatography with an Ultron NC18 column (4.6 by150 mm) (Shinwa Kako, Kyoto, Japan) by the method ofGil-Av et al. (16). Spectrophotometric measurements wereperformed with a Shimadzu UV 3000 recording spectropho-tometer and a Shimadzu CS 900 dual-wavelength TLS scan-ner for gel scannings.

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GC-MS was conducted with a Hewlett-Packard 599 2BGC-MS as follows. Amino acids were derivatized to N-acetyl methyl ester by a modification of the method ofJonsson et al. (22). Selenol and thiol groups ofselenocysteine and cysteine were methylated with methyliodide as described previously (13) before derivatization toN-acetyl methyl ester. GC was run on a capillary column(0.25 mm by 25 m) (model OV101, Gasukuro Kogyo Inc.,Tokyo, Japan). Helium was used as a carrier gas. Thecolumn was programmed from 150 to 220°C at a rate of10°C/min. Ionization voltage was 70 eV. Radioactivity wasmeasured with a Packard Tri-Carb 3320 liquid scintillationspectrophotometer with the toluene system of Bray (4).

Purification of selenocysteine Il-lyase. All operations wereperformed at 0 to 5°C unless otherwise stated. Potassiumphosphate buffer (10 mM) (pH 7.2) containing 0.1 mMphenylmethylsulfonyl fluoride, 20 FM pyridoxal-P, and 0.1%P-mercaptoethanol was used as the standard buffer (bufferA). Buffer B was identical to buffer A, except that 1 mMdithiothreitol was substituted for ,B-mercaptoethanol and 1 Msucrose was added. After each step, the enzyme solutionwas concentrated with an Amicon 200 ultrafiltration unit(Amicon Co., Lexington, Mass.). All dialyses were per-formed with seamless cellulose bags at 4°C for at least 4 h.

(i) Step 1. The cells of C. freundii (ICR 0070) were grownin 330 liters of the complete medium (6), harvested bycentrifugation at the end of the log phase (ca. 12 h), andwashed twice with 0.85% NaCl. The washed cells (2.5 kg[wet weight]) were suspended in 2 liters of buffer A contain-ing 1 mM phenylmethylsulfonyl fluoride and disrupted con-tinuously with a Dynomill (Willy A., Bachofen, Switzerland)at a flow rate of ca. 1 liter/h. After centrifugation at 10,000 xg for 30 min, the supernatant solution was treated withRNase and DNase (0.1 mg/liter each) at room temperaturefor 1 h, followed by centrifugation at 10,000 x g for 1 h. Theclear supernatant solution was dialyzed against four changesof 20 liters of buffer A.

(ii) Step 2. The dialyzed solution was applied to a DEAE-Toyopearl 650M column (17 by 70 cm) equilibrated withbuffer A. After the column was washed with 25 liters of thesame buffer containing 0.1 M KCI, the enzyme was elutedwith 20 liters of the buffer supplemented with 0.12 M KCI ata flow rate of 0.7 liter/h. The active fractions were combined,concentrated, and dialyzed against 100 volumes of buffer A.

(iii) Step 3. The enzyme solution was applied to two phenylSepharose columns (5 by 30 cm) equilibrated with buffer Acontaining 0.5 M phosphate buffer. After successive wash-ings with buffer A containing 0.5, 0.3, 0.2, and 0.15 Mphosphate buffer, the enzyme was eluted with buffer Acontaining 0.1 M phosphate at a flow rate of 0.2 liter/h. Theactive fractions were concentrated and dialyzed against 100volumes of buffer A.

(iv) Step 4. The enzyme solution was applied to a DEAE-Sephadex A50 column (4 by 25 cm) equilibrated with bufferA in which potassium phosphate buffer was replaced byTris-hydrochloride buffer (pH 8.0). The enzyme did not bindto the resin, and the unadsorbed fractions were concentratedand dialyzed as described above.

(v) Step 5. The enzyme solution was applied to ahydroxyapatite column (4 by 10 cm) equilibrated with bufferA. After the column was washed with buffer A containing 20mM phosphate buffer, the enzyme was eluted with buffer Acontaining 30 mM phosphate buffer at a flow rate of 20 ml/hand dialyzed against 100 volumes of buffer B.

(vi) Step 6. The enzyme solution was applied to a Sepha-dex G-150 column (2 by 85 cm) equilibrated with buffer B

TABLE 1. Purification of selenocysteine P-lyase from C. freundiiTotal Total S c ilStep no. (prepn) protein activity (SU/Mct) Yeld(mg) (U) (/g %

1 (Crude extract) 102,000 860 0.008 1002 (DEAE-Toyopearl) 13,500 315 0.023 363 (Phenyl Sepharose) 2,800 172 0.061 204 (DEAE-Sephadex) 1,070 122 0.114 145 (Hydroxyapatite) 48 27 0.56 3.16 (Sephadex G150) 17 16 0.97 1.87 (Toyo Soda TSK DEAE-

3W) 4 16 4.0 1.88 (FPLC0 MonoQ NR 5/5) 0.06 0.39 6.47 0.045

a FPLC, Pharmacia Fast Protein Liquid Chromatography system.

and eluted with the same buffer at a flow rate of 20 mI/h. Theactive fractions were pooled and concentrated.

(vii) Step 7. The resulting enzyme solution was thenchromatographed on a Toyo Soda TSK DEAE-3SWprepacked column equilibrated with buffer B. After washingwith buffer B containing KCI (up to 220 mM), the enzymewas eluted with buffer B containing 240 mM KCI anddialyzed against 100 volumes of buffer B.

(viii) Step 8. The dialyzed enzyme was chromatographedon a MonoQ HR 5/5 anion-exchange column of thePharmacia Fast Protein Liquid Chromatography (FPLC)system. At this step, Tris-hydrochloride buffer (pH 7.4) wassubstituted for potassium phosphate buffer in buffer B. Theelution was carried out with a linear gradient of KCI (0.1 to0.12 M) in buffer B at a flow rate of 1 mllmin. The activefractions were pooled and dialyzed against 100 volumes ofbuffer B.

RESULTSIntracellular localization. We studied the intracellular lo-

calization of selenocysteine ,-lyase in C. freundii cellsessentially by the method of Kaback (23). The specificactivity of the enzyme was 0.43 and 22 mU/mg in themembrane and supernatant fractions, respectively. The en-zyme is located in the cytoplasm.

Purifiation of enzyme. The purification of the enzymeresulted in a ca. 800-fold enhancement of specific activity.Table 1 shows a typical result of purification of the enzyme.The low yield of the enzyme is probably due to inactivationduring purification, because the enzyme appeared to be verylabile at every stage of the purification. We have devised a

way to accomplish purification in a minimum period of time.In particular, the first three steps were performed to reduceas quickly as possible the volume of enzyme solution. Weused ultrafiltration to concentrate the enzyme throughoutpurification, because the fractionation and concentration byammonium sulfate led to the irreversible inactivation of theenzyme. The presence of 0.1% I-mercaptoethanol or 1 mMdithiothreitol was essential to prevent enzyme inactivation.The addition of a high concentration of sucrose (typically 1M) was also found to be effective. However, even with abuffer containing 1 M sucrose, 1 mM dithiothreitol, 50 p.Mpyridoxal-P, and 0.1 mM EDTA as a solvent of enzyme,enzyme activity was decreased gradually during storage.More than 60% of the original activity was lost after storageat -20°C for 1 month.The results of purification might suggest that the purified

enzyme differs from the native enzyme in properties, but wecould not detect any alteration of the kinetic parameters of

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TABLE 2. Amino acid composition of the selenocysteine ,B-lyasefrom C. freundii and pig liver

Amino acid composition of:

Amino acid C. freundii enzyme Pig liver enzymea

MM Mol/moli MO Mol/molbMol% of subunit Mol% of subunit

Aspartic acid 14.12 82 8.15 36Threonine 7.53 43 5.55 25Serine 12.44 72 5.45 24Glutamic acid 6.30 36 11.84 52Proline 3.82 22 5.68 25Glycine 9.99 58 9.06 40Alanine 5.93 34 10.66 47Cysteine 1.05 6 0.84 4Valine 2.47 14 7.58 33Methionine 1.63 9 2.26 10Isoleucine 2.03 12 3.30 15Leucine 4.05 23 9.48 42Tyrosine 4.53 26 1.72 8Phenylalanine 2.69 16 2.82 13Lysine 7.30 42 3.20 14Histidine 4.51 26 2.95 13Arginine 7.89 46 7.19 32Tryptophan (1.73) (10)d 2.98 7C

a Pig liver enzyme was purified as described in reference 12.b Mol/mol values were calculated on the basis of a subunit MW of 64,000

and 48,000 for the bacterial and mammalian enzyme, respectively.c The tryptophan content was estimated spectrophotometrically by the

method of Edelhoch (11). The tyrosine content estimated by this methodagreed well with the amino acid analysis value.

d The low amount of C. freundii enzyme did not allow the spectrophotomet-ric determination of the tryptophan content, and a value of 10 was chosen toallow further calculation.

the enzyme. The fresh crude enzyme showed the same Kmvalue for L- and DL-selenocysteine as did the purified en-zyme (see below and Table 3). Both the purified and crudeenzyme preparations showed the same optimum pH, i.e.,7.0. The purity of the purified enzyme was estimated bypolyacrylamide gel electrophoresis to be at least 95% (asestimated by gel scanning). Above the major protein band, afaint one was detected on the gel. However, when the gelwas stained for selenocysteine P-lyase activity as describedpreviously (12), a singe activity band was observed whichcorresponded to the major protein band. This band corre-sponded also to the single activity band observed with thecrude cell extract.

Physical properties. The MW of the enzyme was estimatedto be 63,000 ± 3,000 by the high-performance gel permeationmethod. Polyacrylamide gel electrophoresis in sodium laurylsulfate gave a major band that had an estimated MW of64,000 ± 1,000. These results suggest that the enzyme iscomposed of a single polypeptide chain. The isoelectricpoint of the protein was estimated to be 6.6 ± 0.1. Theenzyme showed maximum reactivity at ca. pH 7.0 whenassayed in potassium phosphate (pH 5.5 to 9.0) and sodiumborate (pH 7.0 to 9.0) buffers.Amino acid composition. The amino acid compositions of

the C. freundii and pig liver enzymes are summarized inTable 2. A striking feature of the bacterial enzyme is itsrelatively low content of hydrophobic residues (alanine,valine, methionine, isoleucine, leucine, and phenylalanine)(18.8 and 36.1% for the bacterial and mammalian enzymes,respectively). The amino acid compositions of the twoenzymes are fairly different, and no estimation of the degreeof homology in the amino acid sequences can be made.

Cofactor. The absorption spectrum of the enzyme (pH 7.2)

exhibited maxima at 278 and 420 nm, which is characteristicof pyridoxal-P-dependent enzymes. When the enzyme wastreated with 10 mM NH2OH (12), the activity decreased toless than 1%. The activity was 79% (restored by the additionof 50 ,uM pyridoxal-P). Pyridoxal and pyridoxamine 5'-phosphate could not substitute for pyridoxal-P. The Kmvalue for pyridoxal-P was determined to be 0.25 ,uM. Reduc-tion with NaBH4 resulted in the irreversible inactivation ofthe enzyme. Thus, the enzyme requires pyridoxal-P as acofactor, and the coenzyme probably binds to an aminogroup of the enzyme through an aldimine linkage as in otherpyridoxal-P-dependent enzymes studied thus far (10).The cofactor bound to the enzyme was estimated

spectrophotometrically with a molar absorption coefficientof 7,740 M-1 cm-' at 420 nm (pH 7.2), the mean value ofseveral reported ones (27). An average pyridoxal-P contentof 0.8 mol/64,000 g of enzyme was obtained; 1 mol ofpyridoxal-P is thus bound per mol of enzyme.

Substrate specificity. The enzyme catalyzes exclusively the3 elimination of L-selenocysteine to produce L-alanine andelemental selenium. The following selenocysteine analogswere inert: L-cysteine, DL-serine, DL-serine-O-phosphate,L-cysteic acid, glycine, selenium ethyl-L-selenocysteine,S-benzyl-L-cysteine, cysteamine, selenocysteamine, L-homocysteine, L-selenohomocysteine, L-methionine, L-cystathionine, L-selenocystathionine, L-norvaline, and L-selenocysteine ethyl ester. In a reaction system in whichdithiothreitol was omitted, no enzymatic cleavage of DL-selenocystine or DL-cystine was observed. The purifiedenzyme is completely devoid of the following activities:cystathionine P-synthase, alanine transaminase, 0-acetylserine (thiol)-lyase, kynureninase, and aspartate 3-decarboxylase. The latter two catalyze a reaction similar tothe selenocysteine ,B-lyase reaction. When the reaction mix-ture containing DL-selenocysteine was incubated with anexcess of enzyme and the unreacted selenocysteine wasanalyzed by high-pressure liquid chromatography after sele-nium ethylation (13), the amount of D-selenocysteine wasnot changed during the enzyme reaction. This shows that theenzyme does not act on D-selenocysteine and is strictlyspecific for the L-isomer.

Identification of the reaction products. Alanine producedfrom selenocysteine was identified by amino acid analysisand reverse-phase chromatography. It was oxidized quanti-tatively with alanine dehydrogenase, which specifically actson L-alanine.When the reaction was carried out with the standard

reaction mixture in a Thunberg tube (12), the 5,5'-dithio-bis-(2-nitro-benzoic acid) solution in the head compartment wascolored yellow, indicating the formation of volatile H2Se.The production of H2Se was also confirmed by the formationof brownish colloid with lead acetate under acidic conditions(12).To determine whether the actual product of the enzymatic

decomposition of selenocysteine is elemental selenium orH2Se, the reaction was carried out in a reaction mixturedevoid of dithiothreitol (see above). In this reaction system,no H2Se was evolved, although the formation of red elemen-tal selenium was observed and alanine production could beconfirmed with alanine dehydrogenase. The amount of ele-mental selenium formed (700 nmol) was essentially equiva-lent to the amounts of selenocysteine consumed (920 nmol)and alanine produced (830 nmol). These results indicate thatthe selenocysteine 3-lyase catalyzes the removal of elemen-tal selenium from L-selenocysteine and that the formation ofH2Se is due to a nonenzymatic reduction of elemental

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selenium by the excess dithiothreitol present in the reactionmixture.

Isotopic effect. The Km and V,m, values for L- and DL-selenocysteine were found to be similar (Table 3), indicatingthat D-selenocysteine hardly interferes with the reaction ofthe L-isomer. Thus, we have compared the kinetic parame-ters for DL-[a-1H]- and DL-[a-2H]se1enocysteine. The ob-served deuterium isotope effect at the a position ofselenocysteine shows that an a-hydrogen release occurs andis a rate-limiting step in the enzyme reaction.

Reaction in 2H20; When the selenocysteine P-lyase reac-tion was carried out in 2H20, the nuclear magnetic resonancespectrum of alanine produced showed a singlet peak corre-sponding to the a-protons but no detectable peak corre-sponding to the a-proton. The GC-MS analysis of theproduct indicated a mean incorporation' of 3.1 2H atoms permolecule. However, when this sample was analyzed afterracemization in water, the IH-nuclear magnetic resonancespectrum showed a multiplet and a doublet peak correspond-ing to the a- and ,3-proton, respectively. The 2H content wasestimated to be 2.1 per molecule by GC-MS. Amino acidracemase with low substrate specificity acts on alanine andexchanges only the a-proton with 2H in 2H20 (30). There-fore, these results indicate that alanine produced contains 1and 2.1 2H atoms at the a and 1 positions, respectively(Table 4). As unreacted L-selenocysteine or L-alanine incu-bated under the same conditions showed no incorporation of2H, the incorporation of 2H into L-alanine occurred duringthe selenocysteine 1-lyase reaction. When two competitiveinhibitors of the enzyme reaction, L-cysteine and L-cysteinemethyl ester (see below), were incubated under the sameconditions, 2H incorporation was observed in the formercompound only.

Reaction with DL-kx-3H]selenocysteine. To determinewhether a part of the proton abstracted from the a positionof the substrate is returned to the product, the reaction wascarried out with tritiated'substrate, and the incorporation oftritium in the product was monitored. To avoid extensiveexchange of the a-proton with the solvent proton, thereaction was carried out in D20. The isolated L-alanine (0.7p.mol) clearly showed tritium incorporation (Fig. 1); itsspecific radioactivity, however, was low (8,000 dpm/mmol,i.e., 1.5% of that of the original L-selenocysteine). Most ofthe radioactivity of the isolated alanine was lost to thesolvent upon racemization in water. This shows that morethan 90o of the tritium was located at the a position.

Inhibitors. The enzyme was strongly inhibited by 1 mM ofthe following metallic dications: Zn, Ni, Pb, and Hg. It wascompletely inactivated by incubation at 37°C for 3 h with 1mM of thiol reagents such as iodoacetic acid, iodoacetamide,and N-ethylmaleimide. However, D-cycloserine and L-propargylglycine were inert.

L-Cysteine and L-Cysteine methyl ester were found tobehave as competitive inhibitors against L-selenocysteine

TABLE 3. Kinetic isotopic effect with [a-2H]selenocysteine

Substrate Km (mM) V.., (gmol)min Vnax/K.L-Selenocysteine 0.95 9.49 9.99DL-Selenocysteinea 0.98 9.04 9.22DL-[a-2HIselenocysteinea 0.98 5.79 5.91

Isotope effect 1.56 1.56

a The Km values were calculated for the L-isomers.

TABLE 4. Selenocysteine ,3-lyase reactiona in D20

No. of 2Hincorporatedb

Substrate Analyzed compound at position:

Selenocysteine Selenocysteine 0 0Selenocysteine Alanine 1 2.1Alanine Alanine 0 0Cysteine Cysteine 0.74 0Cysteine mnethyl ester Cysteine methyl ester 0 0

a Incubation conditions are described in the text. All incubated compoundswere L-isomers.

b The incorporation and distribution of 2H were determined by 'H-nuclearmagnetic resonance and by GC-MS, as indicated in the text.

(Ki, 0.65 and 0.6 mM, respectively), even in the presence ofa large excess (0.1 mM) of pyridoxal-P. When L-cysteinemethyl ester was incubated without enzyme for 2 h under theassay conditions and the mixture was analyzed by thin-layerchromatography with a solvent system in' which L-cysteinemethyl ester (Rf, 0.83), cysteine (Rf, 0.40), and cystine (Rf,0.05) were clearly separated (methanol-acetone, 1:1[vol/vol]), no appreciable amounts of cystine and cysteinewere detected. This indicates that the inhibition cannot beattributed to L-cysteine that may arise from the ester bynonenzymatic hydrolysis but is intrinsically due to L-cysteine methyl ester.None of the following compounds inhibited the enzyme

reaction with 4 mM DL-selenocysteine: 4 mM L-seleno-cysteine ethyl ester, L-aspartate, glycine, L-alanine, L-nor-leucine, DL-serine, and hydrogen selenide. Neither D-cysteine nor cysteamine inhibited the enzyme reaction at 5mM when sufficient pyridoxal-P (0.1 mM) was present in theassay mixture.

Reactions with 0-chloro-L-alanine. When L-Cl-alanine wasincubated with the enzyme under the reaction conditions for

150

00100 I &

Ea0

50

01 2 3 4 5 6 7 8 9 10

Fraction NumberFIG. 1. Analysis by thin-layer chromatography of alanine pro-

duced from DL-[a-3H]selenocysteine. A portion of the sample (ca. 75p.mol, 900 dpm) was chromatographed as described in the text withparallel runs of authentic alanine and selenocysteine. Five-millimeter strips of the plate were cut and dipped in 200 ,ul of waterfor 2 h, and the radioactivities were measured.

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674 CHOCAT ET AL.

\N.~~~~~~~~~~~~~~~.

0 ~~20

06A50 1 2 10

10

TI)E 1/CIAIa (mM)

0

~~~~~~~~~0z

w

10

0 10 20 30 40

TIME (min)FIG. 2. Inactivation of selenocysteine P-lyase with L-Cl-alanine.

The enzyme was preincubated with various concentrations of L-Cl-alanine (none [* , 0.71 mM [0], 1.25 mM [A], and 2.5 mM [C]),and the remaining activity was assayed as described in the text. Theinset shows a double-reciprocal plot of the apparent first-order rateconstants for the inactivation versus L-Cl-alanine concentrations.

selenocysteine, ammonia, pyruvate, and Cl- were formed.Pyruvate was converted quantitatively to alanine with ala-nine dehydrogenase and identified by amino acid analysis.Ammonia and chloride anions were identified by amino acidanalysis and the method of Iwasaki (20), respectively. Bal-ance studies of the a,, elimination showed that L-Cl-alanineis converted into equimolar amounts of pyruvate and chlo-ride anions. The kinetic parameters for L-Cl-alanine were asfollows: Km - 3.1 mM and Vma, = 2.94 ixmol min-' mg-1.This reaction was inhibited by L-selenocysteine (Ki = 0.84mM) and L-cysteine, but not by L-alanine, L-methionine,L-lysine, L-serine, L-tyrosine, L-histidine, or D-cysteine.Furthermore, L-Cl-alanine inhibits the ,B elimination ofselenocysteine in a competitive manner; the Ki value (2.5mM) for L-Cl-alanine is substantially consistent with its Kmvalue in the a,P elimination reaction. These results suggestthat both reactions are catalyzed by the same enzyme and atthe same site.When the purified enzyme was preincubated with L-Cl-

alanine as described above, a time-dependent inactivation ofthe enzyme was observed. However, the apoenzyme wasnot affected by L-Cl-alanine. This suggests that the inactiva-tion is based on enzyme catalysis. The inactivation appearsto be irreversible, since no activity was recovered upondialysis of the inactivated enzyme against 10 mM buffer B for4 h. Typical semilogarithmic plots of the remaining activityversus time are shown in Fig. 2. From a double-reciprocalplot of the apparent first-order rate constants of inactivationversus L-Cl-alanine concentrations, the apparent Kinac valueand the maximal inactivation rate constant were calculatedto be 3.1 mM and 0.23 min-', respectively.

The binding constant obtained was in good agreement withthe Km value (3.1 mM) for L-Cl-alanine in the a, eliminationreaction. The inactivation by L-Cl-alanine was almost com-pletely prevented by the presence of 3 mM L-selenocysteineor L-cysteine in the incubation mixture. However, none ofthe following was effective: 3 mM L-methionine, L-lysine,L-alanine, L-serine, L-tryptophan, L-histidine, or D-cysteine.A double-reciprocal plot of the apparent first-order rateconstants at a given concentration of L-Cl-alanine (1.8 mM)versus L-selenocysteine concentrations gave a straight line.The apparent Ki value of L-selenocysteine for the protectionof the enzyme against inactivation was calculated to be 0.73mM, which is close to the Km value of L-selenocysteine forthe ,B elimination reaction. These results suggest that theinactivation proceeds via a saturable enzyme-inhibitor com-plex and that this complex is a common intermediate in thea43 elimination and inactivation reactions. Based on anenzyme MW of 64,000, the partition ratio between a,,Belimination and inactivation is 825.

Inactivation of selenocysteine ,-lyase by incubation withL-selenocysteine. When the enzyme was assayed with L-selenocysteine in the absence of added pyridoxal-P (lessthan 0.1 ,uM), the enzyme reaction initially proceededlinearly with time and then slowed down (Fig. 3). Thereaction rate was restored by a subsequent addition ofpyridoxal-P (at a final concentration of 0.1 mM). When theenzyme was preincubated in the absence of added pyridoxal-P with 5 mM L-selenocysteine, L-cysteine, L-aspartate,L-alanine, L-serine, L-methionine, or L-lysine in 10 mMbuffer B at 37°C for 2 h and the remaining activity wasmeasured, inactivation occurred specifically in the presenceof L-selenocysteine. Therefore, this excludes the possibilityof a simple dilution of the cofactor in the assay mixture asthe pyridoxal-P concentration becomes lower than its Km.

0.2 Pyridoxal-P

w

0O.1 7-fPL0

0 , ,

co

0 60 120

TIME (nm)FIG. 3. Effect of pyridoxal-P on selenocysteine P-lyase reaction.

The reaction was carried out in the presence (+PLP) or absence(-PLP) of pyridoxal-P (0.1 ,umol) in 1 ml of the standard reactionmixture, and the production of L-alanine was followedspectrophotometrically with alanine dehydrogenase in the presenceof NAD. At the point indicated by the arrow, pyridoxal-P (finalconcentration, 0.1 mM) was added.

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SELENOCYSTEINE 3-LYASE FROM C. FREUNDII 675

L-Cysteine (1 mM) (but not L-aspartate, L-alanine, or L-serine) protected the enzyme from inactivation by L-selenocysteine (Table 5). This specific protective effect by acompetitive inhibitor suggests that inactivation occurs dur-ing the normal processing of L-selenocysteine. Inactivationcould also be prevented by the addition into the preincuba-tion mixture of the following a-keto acids: pyruvate, a-ketoglutarate, and glyoxylate (Table 5).

DISCUSSIONSelenocysteine 13-lyase from C. freundii is strikingly dif-

ferent from the pig liver enzyme reported previously (12) inits physicochemical properties (subunit structure, isoelectricpoint, number of cofactor molecules per subunit, and aminoacid composition). However, both enzymes are very similarin their enzymological properties, i.e., both exhibit verystrict specificity for L-selenocysteine, similar Km values fortheir substrates, and competitive inhibition by L-cysteine.Inactivations by L-Cl-alanine and L-selenocysteine were alsoobserved with the mammalian enzyme.The absolute requirement for pyridoxal-P of the enzyme

activity and the deuterium isotope effect observed at the aposition of the substrate suggest that the 1 eliminationreaction proceeds as has been proposed for aspartate -decarboxylase (5) or mammalian selenocysteine 13-lyase(Esaki et al., in press) (Fig. 4). The isotope effect observedwith DL-[a-2H]selenocysteine suggests that the reaction pro-ceeds via the initial labilization of a-proton to produce aquinoid intermediate designated as (II). When the reactionwas carried out in 2H20, in addition to the incorporation ofone 2H atom into the 1B position of alanine after the removalof elemental selenium, an average of 1.1 of the two 13-hydrogen atoms of selenocysteine was exchanged with thesolvent 2H. This is consistent with a rapid reversible reactionbetween intermediate (III) and (IV). The internal return ofthe proton to the a position of alanine as observed withtritiated selenocysteine suggests that the a deprotonationand protonation are performed by a single base of theenzyme. Protonation at the 1 position of the enzyme isprobably catalyzed by another base of the enzyme. Such atwo-base mechanism has been proposed for the mammalianenzyme (Esaki et al., in press) and bacterial aspartate1-decarboxylase (5).Although not a substrate, L-cysteine is a competitive

inhibitor of the enzyme. The incorporation of 2H into L-cysteine upon incubation with the enzyme in 2H20 suggests

TABLE 5. Protective effect of various compounds against theinactivation of the enzyme by preincubation with L-

selenocysteineaAmino acid or Residual activitya-keto acid

None ................................... 11L-Cysteine ................................. 70L-Aspartate................................. 15L-Alanine .................................. 8L-Serine................................... 11Pyruvate ................................... 62a-Ketoglutarate ............................. 56Glyoxylate ................................. 70a-Ketoisocaproate .......................... 35

a The preincubation mixture contained 3 p.mol of sodium borate buffer (pH7.2), 60 nmol of L-selenocystine, 0.3 p.mol of dithiothreitol, the indicatedamino acid (30 nmol) or a-keto acid (150 nmol), and 0.025 U of enzyme in afinal volume of 30 p1. After incubation for 2 h at 37°C, the residual activity wasassayed as indicated in the text.

H HCoO

(IV)

- Inactivated

Enzyme

Pyruvate

H(V)

Pyridoxal-P+

Alanine

FIG. 4. Proposed reaction mechanism of selenocysteine ,B-lyase.X, Cl or Se- (as selenohydryl groups are essentially in a dissociatedform under the experimental conditions).

the abortive formation of a cysteine-pyridoxal intermediateanalogous to (II). However, L-cysteine methyl ester does notincorporate 2H under the same conditions, despite its stronginhibition of the selenocysteine ,B-lyase reaction. This meansthat the reaction does not proceed up to the formation of theL-cysteine methyl ester analog of (II) and that the competi-tive inhibition could be better explained by the formation ofan L-cysteine methyl ester-pyridoxal-P thiazolidine deriva-tive at the active site of the enzyme.The inactivation of the enzyme during the reaction course

in the absence of added pyridoxal-P was found to (i) occurspecifically with L-selenocysteine, (ii) be prevented by acompetitive inhibitor (L-cysteine) of the enzyme, and (iii) bereversed by various a-keto acids. Although we have littleevidence, these results suggest that the enzyme is inacti-vated through the accidental formation of enzyme-boundpyridoxamine 5'-phosphate cysteine, as has been proposedfor the mammalian enzyme (Esaki et al., in press). Thetransamination probably occurs by the erroneousprotonation at the C-4' of the ketimine-quinoid intermediate(II or IV) either before or after removal of the seleniumatom; the determination of the exact reaction mechanismawaits the identification of the actual reaction product (1-selenohydrylpyruvate or pyruvate [8]). The ratio of the 13elimination and transamination reactions was calculated tobe 40,000 in the case of the C. freundii enzyme. Thisexcludes the possibility that the enzyme may function in thecells as a transaminase. However, we cannot make anyhypothesis about the significance in vivo of this reaction forthe bacterial enzyme.The inactivation of selenocysteine 1-lyase by L-Cl-alanine

exhibits the characteristics of suicide inactivation; i.e., pseu-do-first-order kinetics, irreversibility, and protection by thesubstrate. Although the actual form of the inactivated en-

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676 CHOCAT ET AL.

zyme is not known, a possible mechanism for the inactiva-tion, based on models proposed in the literature (24, 34), isalso represented in Fig. 4. The relatively low partition ratio,825, of the a,4 elimination reaction to the inactivationreaction is similar to that reported by Walsh et al. withalanine racemase (34) and indicates the high efficiency of thealkylating reaction. These results exclude the possibility thatthe enzyme may catalyze in vivo the a,1 elimination of anysubstrate, because this reaction would proceed through thesame a-aminoacrylate intermediate to result in the rapidinactivation of the enzyme.

Thus, the only reaction likely to be catalyzed in vivo bythe enzyme is the B elimination of an L-amino acid (assuggested by the fact that selenocysteamine is inert) with afree carboxyl group and'a good electrophilic leaving group atthe P position. Such naturally occurring compounds, otherthan selenocysteine, are very few. L-Aspartate and L-kynurenine have such characteristics but were inert as asubstrate.A plausible supposition for the physiological role of the

enzyme could be the detoxification of L-selenocysteine toelemental selenium. L-Selenocysteine was found to inhibitthe growth of various aerobic bacterial strains on sulfur-limited medium at the 0.1-ppm (wt/vol) level (unpublisheddata); this compound appears to be very toxic. However, wewere unable to find any change in the enzyme specificactivity when the cells were grown in media containingvarious sources of carbon, sulfur, or selenium at variousconcentrations (unpublished data). Moreover, very little isknown about the status (Se' or H2Se), toxicity, and eventualmetabolism of elemental selenium in bacterial cells.

LITERATURE CITED1. Abeles, R., and C. Walsh. 1973. Acetylenic enzyme inactivators.

Inactivation of -y-cystathionase, in vitro and in vivo, bypropargylglycine. J. Am. Chem. Soc. 95:6124-6125.

2. Biemann, K., J. Seibi, and F. Gapp. 1961. Mass spectra oforganic molecules: ethyl ester of amino acids. J. Am. Chem.Soc. 83:3795-3804.

3. Braunsteim, A. E., E. W. Goryachenkova, and N. D. Lac. 1969.Reactions catalyzed by serine sulfhydrylase from chicken liver.Biochim. Biophys. Acta 171:366-368.

4. Bray, G. A. 1960. A simple efficient liquid scintillator forcounting aqueous solutions in a liquid scintillation counter.Anal. Biochem. 1:279-285.

5. Chang, C. C., A. Laghai, M. H. O'Leary, and H. G. Floss. 1982.Some stereochemical features of aspartate 0-decarboxylase. J.Biol. Chem. 257:3564-3569.

6. Chocat, P., N. Esaki, T. Nakamura, H. Tanaka, and K. Soda.1983. Microbial distribution of selenocysteine lyase. J. Bacte-riol. 156:455-457.

7. Cone, J. E., R. Martin del Rio, J. N. Davis, and T. C. Stadtman.1976. Chemical characterization of the selenoprotein compo-nent of clostridial glycine reductase: identification ofselenocysteine as the organoselenium moiety. Proc. Natl. Acad.Sci. U.S.A. 73:2659-2663.

8. Cotton, F. A., and G. Wilkinson. 1976. Basic inorganic chemis-try. John Wiley & Sons, Inc., New York.

9. Davis, B. J. 1964. Disc electrophoresis. II. Methods and appli-cation to human serum proteins. Ann. N.Y. Acad. Sci.121:404 427.

10. Davis, L., and D. E. Metzler. 1972. Pyridoxal-linked eliminationand replacement reactions, p. 33-74. In P. D. Boyer (ed.), Theenzymes, vol. 7. Academic Press, Inc., New York.

11. Edelhoch, H. 1967. Spectrophotometric determination oftryptophan and tyrosine in proteins. Biochemistry 6:1948-1954.

12. Esaki, N., T. Nakamura, H. Tanaka, and K. Soda. 1982.Selenocysteine lyase, a novel enzyme that specifically acts onselenocysteine: mammalian distribution and purification and

properties of pig liver enzyme. J. Biol. Chem. 257:4386-4391.13. Esaki, N., T. Nakamura, H. Tanaka, T. Suzuki, Y. Morino, and

K. Soda. 1981. Enzymatic synthesis of selenocysteine in ratliver. Biochemistry 20:4492-4496.

14. Forstrom, J. W., J. J. Zakowsky, and A. L. Tappel. 1978.Identification of the catalytic site of rat liver glutathione perox-idase as selenocysteine. Biochemistry 17:2639-2644.

15. Gaitonde, M. K. 1967. A spectrophotometric method for thedirect determination of cysteine in the presence of other natu-rally occuring amino acids. Biochem. J. 104:627-633.

16. Gil Av, E., A. Tishbee, and P. E. Hare. 1980. Resolution ofunderivatized amino acids by reverse-phase chromatography. J.Am. Chem. Soc. 73:2659-2663.

17. Gunther, W. H. H. 1967. Methods in selenium chemistry. III.The reduction of diselenides with dithiothreitol. J. Org. Chem.32:3931-3933,

18. Hartmanis, M. G. N., and T. C. Stadtman. 1982. Isolation of aselenium-containing thiolase from Clostridium kluyveri: identi-fication of the selenium moiety as selenomethionine. Proc. Natl.Acad. Sci. U.S.A. 79:4912-4916.

19. Inagaki, K., K. Tanizawa, H. Tanaka, and K. Soda. 1984.Purification and properties of amino acid racemase from Aero-monas punctata subsp. caviae, p. 355-363. In A. E.Evangelopoulos (ed.), Chemical and biological aspects of vita.min B6 catalysis. Alan R. Liss, Inc., New York.

20. Iwasaki, I., S. Utsumi, K. Hagino, and T. Ozawa. 1956. A newspectrophotometric method for the determination of smallamounts of chloride using the mercuric thiocyanate method.Bull. Chem. Soc. Jpn. 29:860-864.

21. Jones, J. B., G. L. Dilworth, and T. C. Stadtman. 1979.Occurrence of selenocysteine in selenium-dependent formatedehydrogenase of Methanococcus vannielii. Arch. Biophys.Biochem. 195:255-260.

22. Jonsson, J., J. Eyem, and J. Sjoquist. 1973. Quantitative gaschromatography of amino acids on a short glass capillarycolumn. Anal. Biochem. 51:204-219.

23. Kaback, H. R. 1971. Bacterial membranes. Methods Enzymol.22:99-120.

24. Likos, J. J., H. Ueno, R. W. Feldhaus, and D. E. Metzler. 1982.A novel reaction of the coenzyme of glutamate decarboxylasewith L-serine-O-sulfate. Biochemistry 21:4377-4386.

25. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

26. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

27. Morino, Y., and S. Tanase. 1980. Structure and properties ofvitamin B6, p. 159-181. In Committee of the Japanese Associ-ation of Vitaminology (ed.), Vitaminology (II). Tokyo KagakuDojin Ltd, Tokyo, Japan.

28. Ng, B. H,, and J. W. Anderson. 1978. Synthesis of sele-nocysteine by cysteine synthases from selenium accumulatorand non-accumulator plants. Phytochemistry 17:2069-2074.

29. Ohshima, T., and K. Soda. 1979. Purification and properties ofalanine dehydrogenase from Bacillus sphaericus. Eur. J.Biochem. 100:29-39.

30. Sawada, S., N. Esaki, T. Yagi, and K. Soda. 1984. Reactionmechanism of amino acid racemase with low substrate speci-ficity of Pseudomonas striata. Bull. Kyoto Univ. Educ. Ser.Math. Nat. Sci. B 64:21-31.

31. Soda, K. 1968. Microdetermination of D-amino acids and D-amino acid oxidase activity with 3-methyl-2-benzothiazolonehydrazone hydrochloride. Anal. Biochem. 25:228-235.

32. Spackman, D. H., W. IH. Stein, and S. Moore. 1958. Automaticrecording apparatus for use in the chromatography of aminoacids. Anal. Chem. 30:1190-1206.

33. Tiselius, A., S. Hjerten, and 0. Levin. 1956. Protein chromatog-raphy on calcium phosphate columns. Arch. Biochem. Biophys.65:132-155.

34. Wang, E., and C. Walsh. 1978. Suicide substrates for the alanineracemase of Escherichia coli B. Biochemistry 17:1313-1321.

35. Wrigley, C. W. 1971. Gel electrofocusing. Methods Enzymol.22:559-564.

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