J. Biochem. 89, 599-607 (1981)

Purification and Some Properties of Sarcosine Oxidase from Corynebacterium sp. U-96

Masaru SUZUKI

Noda Institute for Scientific Research, Noda, Chiba 278

Received for publication, July 18, 1980

During the course of studies on creatinine metabolism by bacteria, a newly isolated bacterial strain, Corynebacterium sp. U-96, showed the highest potency in the pro duction of sarcosine oxidase. The enzyme was purified from the cell-free extract to a homogeneous state by successive procedures involving polyethyleneimine treatment and chromatographies on DEAE-cellulose, QAE-Sephadex A-50, DEAE Sephadex A-50, Ultrogel AcA34, and creatine-AH-Sepharose 4B. The absorption spectrum of the enzyme had maxima at 276, 369, and 451 nm, being characteristic of a fiavoprotein. This enzyme contains both 1 mol of non-cova lently bound FAD and 1 mol of covalently bound FAD per mol of enzyme. Sedimentation experiments gave an s?o,W value of 9.35 and the molecular weight was calculated to be 174,000 by the meniscus depletion method. The enzyme was shown to be composed of one each of four non-identical subunits; subunit A 110,000; B 44,000; C 21,000; D 10,000. One moleculk of FAD is covalently bound to subunit B.

The enzyme acted preferentially on sarcosine rather than on N-methyl-L-alanine

or N-ethyl-glycine, but did not act at all on other N-methyl-L-amino acids tested. The K„ and kcat values of the enzyme were estimated to be 3.4 mM and 5.8 s-1 for

sarcosine at pH 8.3 and 37•Ž, 8.7 mM 2.1 s-1 for N-methyl-L-alanine at pH 8.3 and

37•Ž, and 11.4 mat and 2.2 s-1 for N-ethyl-glycine at pH 7.0 and 37•Ž, respectively.

The Km and Vmax values for oxygen as a hydrogen acceptor were found to be 0.13 mM and 12.81 ƒÊmol/min/mg of enzyme. Oxygen can be replaced by 2,6

- dichlorophenolindophenol, phenazine methosulfate, ferricyanide, but not by meth

ylene blue or cytochrome c. The enzyme activity was strongly inhibited by Zn2+ Cua+ Cd2+ Hg2+ Ag+ iodoacetamide, p-chloromercuribenzoate, or N-ethylmaleimide. Acetate, propi onate, formaldehyde, and acetaldehyde also had inhibitory effects on the enzyme activity. The stoichiometry data showed that oxygen consumption was accompanied by the formation of equimolar amounts of formaldehyde, glycine, and hydrogen peroxide from sarcosine.

Abbreviations: TCA, trichloroacetic acid; PMS, phenazine methosulfate; 2, 6-DCPI, 2, 6-dichlorophenolindo phenol; p-CMB, p-chloromercuribenzoate.

Vol. 89, No. 2, 1981 599 600 M. SUZUKI

The oxidation of sarcosine in mammalian liver sarcosine, 30 ƒÊmol of glycylglycine buffer, pH 8.3 mitochondria has been considered to be due to and the enzyme in a total volume of 0.5 ml was sarcosine dehydrogenase (sarcosine: (acceptor) oxi incubated for 10 min at 37•Ž. The reaction was doreductase (demethylating), EC 1.5.99.1) tightly stopped with 0.5 ml of 0.5 N acetic acid, followed bound to an electron transferring flavoprotein by mixing with 3 ml of a color reagent (10 (ETF) (1). Therefore, purified liver sarcosine dibasic ammonium phosphate and 0.02% acetyl dehydrogenase free from ETF could no longer acetone pH 6.5). The mixture was incubated for oxidize sarcosine in the absence of artificial hydro 40 min at 37•Ž, then its color intensity was read gen acceptors such as indophenol and PMS (2). at 410 nm. Blanks lacking enzyme were run with Oka et al. (3) showed that sarcosine dehydro each batch of assays. One unit of sarcosine genate from Pseudomonas putida also requires oxidase activity was defined as the amount of electron carriers for its catalytic action and never enzyme which produces 1 ƒÊmol of formaldehyde

produces hydrogen peroxide. per min under the above conditions. The activity Recently the author found another type of was proportional to the enzyme concentration enzyme (sarcosine oxidase, sarcosine: oxygen oxi unless the change in absorption at 410 rim exceeded doreductase (demethylating), EC 1.5.3.1) in Cory 0.35. Specific activity was defined as activity unit

nebacterium sp. U-96 and purified it to a homo per mg of protein. geneous state. This novel enzyme contains both Determinations-Protein concentration was I mot of non-covalently bound FAD and 1 mol estimated from the absorbance at 280 nm by using of covalently bound FAD per mot of enzyme, and an E1%1cmvalue of 13.1, which was obtained by catalyzes the oxidation of sarcosine without addi absorbancy and dry weight measurements of the tion of a hydrogen acceptor such as PMS or purified enzyme preparation. Sarcosine and gly 2,6-DCPI, producing equimolar amounts of form cine were analyzed by a Hitachi model 835 auto aldehyde, glycine, and hydrogen peroxide in the matic amino acid analyzer. Oxygen consumption presence of oxygen. was determined with a Fieldlab oxygen analyzer, This paper describes the purification and some model 100800 (Beckman Instruments Inc.). AMP characteristics of sarcosine oxidase from Coryne was determined by enzymatic analysis with myo bacterium sp. U-96. kinase (ATP: AMP phosphotransferase EC 2.7.4.3) (6). MATERIALS AND METHODS Materials-AH-Sepharose 4B was purchased from Pharmacia. N-Methyl derivatives of L-alanine,

Microorganism-A bacterial stain was isolated L-valine, L-leucine, L-isoleucine, L-serine, and L

from soil by growth on a medium containing phenylalanine were obtained from Bachem. Inc, creatinine as a sole carbon source, and taxonomi Fine Chemicals. N-ƒÃ-Methyl-L-lysine, N-ethyl

cally identified as Corynebacterium sp. U-96 by glycine, sarcosyl-glycine, and sarcosyl-sarcosine referring to the description of Bergey's Manual were from Vega Fox Biochemicals. Bovine liver

(4). This bacterium was cultivated aerobically for catalase (hydrogen peroxide: hydrogen peroxide 20 h at 37•Ž in a 200-liter tank containing a oxidoreductase, EC 1.11.1.6) was from Sigma, medium, 160 liters, with the following composi Chem. Co., Ltd. Horse radish peroxidase (donor: tion: 0.8% sarcosine, 2.1% yeast extract, 0.05 hydrogen peroxide oxidoreductase, EC 1.11.1.7)

KH2PO4, 0.2% K2HPO4, 0.01%. MgSO4•E7H2O, was from Toyobo Co., Ltd. (Japan) . Pyruvate and 0.005% FeCl3, pH 6.5. The cells (6.4 kg as kinase (ATP : pyruvate 2-o-phosphotransferase, wet weight) were harvested by continuous cen EC 2.7.1,40) and myokinase were from Oriental trifugation at 13,000 •~ g and frozen for storage at Yeast Co., Ltd. (Japan). Lactate dehydrogenase -20•Ž. (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) and

Enzyme Assay-The activity of sarcosine oxi phosphodiesterase of Crotalus adamanteus venom dase was determined by measuring the amount of (oligonucleate 5•Œ-nucleotidohydrolase, EC 3.1.4.1) formaldehyde formed according to the method of were from Sigma Chem. Co., Ltd. The standard

Nash (5) with sarcosine as a substrate. In this protein kit for estimation of molecular weight was system, a reaction mixture containing 60 ƒÊmol of from Boehringer Mannheim, Germany. Other

J. Biochem. BACTERIAL SARCOSINE OXIDASE 601

chemicals used were commercial products. Step 1. Preparation of crude enzyme: Fro Preparation of Creatine-AH-Sepharose 4B zen cell paste (6.4 kg as wet weight) was thawed AH-Sepharose 4B (3 g) was swollen with 600 ml in 30 liters of 0.01 M potassium phosphate buffer , of 0.5 M NaCl and washed with 200 ml of distilled pH 8.0 (Buffer A). The suspension was contin water, pH 4.5. Next, 12 ml of 1 % N-(3-dimethyl uously homogenized with a Dyno Mill KDL aminopropyl)-N-ethylcarbodiimide hydrochloride (Willy A. Bachofen M.E.B., Switzerland). (pH 4.5) was added, and the pH of the solution Step 2. Polyethyleneimine treatment: The was maintained at 6.0 with 0.1 N NaOH . This homogenate (28 liters) was brought to 0.2 M salt coupling solution was mixed with creatine solution concentration by adding solid ammonium sulfate (pH 6.0) to make a final concentration of creatine with mechanical stirring, followed by adding 930 of 1.3% and the mixture was incubated overnight ml of 10% polyethyleneimine (PEI), pH 8.0. The at room temperature with gentle shaking. Crea solution was stirred for 20 min, and the precipitate tine-AH-Sepharose 4B thus prepared was thor was removed by continuous centrifugation at oughly washed with distilled water on a glass filter . 13,000 x g. The supernatant (24 liters) was con The gel was suspended in 0.01 M potassium phos centrated to 4.5 liters with a hollow-fiber concen phate buffer, pH 8.0. trating apparatus (model IL-100, Asahi Kasei Ltd.) Polyacrylamide Gel Electrophoresis-Poly and diluted with 40 liters of Buffer A to reduce acrylamide gel electrophoresis was performed the salt concentration. according to the method of Davis (7) at pH 8.3. Step 3. DEAF-cellulose chromatography: SDS-Polyacrylamide Gel Electrophoresis-The To the diluted solution, 4.8 kg of wet DEAE enzyme was treated at 100•Ž for 5 min in the - cellulose previously equilibrated with Buffer A was presence of 1 % SDS or denatured by treatment added. The mixture was stirred gently for 1 h to with guanidine hydrochloride followed by alkyla absorb the enzyme. After decantation of the tion according to the method of Weber and Os supernatant, DEAE-cellulose which had absorbed born (9). the enzyme was packed into a column (16 x 80 Sedimentation Analysis-Ultracentrifugal sedi cm), washed with 20 liters of Buffer A containing mentation runs were carried out with a Hitachi 0.15 M KCl and eluted with 10 liters of Buffer A UCA-1A analytical ultracentrifuge at 20•Ž and containing 0.4 M KCl. The eluate was collected 55,047 rpm with the bar angle at 70?. The con in 500 ml fractions. The fractions with enzyme centration of the enzyme was changed between activity had a high yellow color.

0.182 and 0.728 % in 10 mM potassium phosphate Step 4. First Ultrogel AcA34 chromatog buffer containing 0.1 M KCl, pH 8.0. raphy: The enzyme solution obtained from the

Hydrogen-Acceptor Specificity-The reduction previous step was concentrated to 500 ml by of various acceptors was followed in a Klett ultrafiltration (model IL-100, Asahi Kasei Ltd.)

Summerson photometer in terms of the absorption and passed through a column (6 •~ 140 cm) of decrease at the appropriate wavelengths. The Ultrogel AcA34 previously equilibrated with Buffer reactions were carried out at 37•Ž under anaerobic A containing 0.1 M KCl at a flow rate of 60 ml conditions. For studying the effect of oxygen per h. The fractions (120 ml) with enzyme activity concentration on the rate of oxidation, the solu were combined. tions were saturated with oxygen or nitrogen to Step 5. Chromatography on QAE-Sephadex give a desired concentration. Km and Vmax were A-50: A part of the pooled active fractions (220 obtained from Lineweaver-Burk plots. ml) was charged on a QAE-Sephadex A-50 column

(3.5 •~ 40 cm) previously equilibrated with Buffer A containing 0.1 M KCl. The column was washed RESULTS with Buffer A containing 0.25 M KCl and eluted

Purification of the Enzyme-The various stages with a linear gradient of KCl from 0.25 to 0.6 M in the purification of sarcosine oxidase from (1 liter of total volume) in Buffer A. The peak of Corynebacterium sp. U-96 are described in detail enzyme activity was eluted between 0.43 and 0.5 M below. All operations were carried out at below KCl.

8•Ž unless otherwise stated. Step 6. Chromatography on DEAE-Sephadex

Vol. 89, No. 2, 1981 602 M. SUZUKI

A-50: The enzyme solution (240 ml) derived from was obtained. By calibration with several standard step 5 was dialyzed against Buffer A containing proteins, their molecular weights were determined 0.1 M KCl overnight in a cold room. The solution to be 110,000, 44,000, 21,000, and 10,000. Den was applied to a column (3.5 x 40 cm) of DEAE sitometric scanning of the stained gel showed that Sephadex A-50 previously equilibrated with Buffer the relative ratio of peak areas was 2.56(A) : A containing 0.25 M KCl. The elution was carried 1.00(B) : 0.45(C) : 0.22(D). Fluorescence was ob out with a linear gradient between 0.25 and 0.6 M served exclusively in subunit B; that is, flavin KCl. binds to this subunit. These results suggest the Step 7. Second Ultrogel AcA34 chromatog enzyme consists of four non-identical subunits. raphy: The enzyme solution was concentrated to The sedimentation pattern of the enzyme 100 ml in an Amicon ultrafiltration apparatus (PM appeared as a single symmetric peak during the 30 membrane) and divided into 20 ml fractions. run (Fig. 3). Each of them was passed through a column (4 x The sedimentation pattern of the enzyme 145 cm) of Ultrogel AcA34 with Buffer A contain appeared as a single symmetric peak during the ing of 0.1 M KCl at a flow rate of 60 ml per h. run (Fig.3). Thesedimentation coefficient (S020,W) Step 8. Chromatography on creatine-AH-Se pharose 4B: The enzyme solution (800 ml) pre pared in step 7 was diluted two-fold with Buffer A and 160 ml fractions were applied to the top of a column (1 •~ 20 cm) of creatine-AH-Sepharose 4B equilibrated with Buffer A, and the column was washed with 100 ml of Buffer A. The enzyme was subsequently sarcosine oxidase reached 8.93 U/mg. A summary of a typical purification pro cedure is presented in Table I. Properties of Enzyme-Purity and molecular weight: Polyacrylamide gel electrophoresis of the purified enzyme showed only one protein band on staining the gel with Amido Black 10B (Fig. 1). Fig. 1. Polyacrylamide gel electrophoresis of purified The enzyme was subjected to SDS-polyacrylamide Corynebacterium sarcosine oxidase. Purified sarcosine gel electrophoresis. As shown in Fig. 2, there oxidase (40 ƒÊg protein) was subjected to electrophoresis were four bands of stained protein, designated on 7.5% gel. The gel was removed from the column tentatively as subunits A, B, C, and D. When after a run at 5 mA per gel at pH 8.3 for 50 min, and the alkylated enzyme was used, the same result stained with 1 % Amido Black 10 B in 7% acetic acid.

TABLE I. Summary of the purification of sarcosine oxidase from Corynebacterium sp. U-96.

J. Biochem. BACTERIAL SARCOSINE OXIDASE 603

Fig. 4. Dependence of the sedimentation coefficient on the concentration of Corynebacterium sarcosine oxidase.

Fig. 2. SDS-polyacrylamide gel electrophoresis of puri fied Corynebacterium sarcosine oxidase. The purified enzyme, 130 ƒÊg, in 1 ml of 0.01 M potassium phosphate,

pH 8.0, containing 25% glycerol, 1% SDS, and 4% 2 - mercaptoethanol, was treated at 100•Ž for 3 min, then 0.02 to 0.1 ml aliquots were applied to 7.5% gels and run at a constant current of 8 mA per gel for 4 h at room temperature. The molecular weight of the enzyme was estimated by comparison with parallel runs of standard marker proteins such as trypsin inhibitor, bovine serum albumin, and RNA-polymerase (subunits ƒ¿, ƒÀ, and ƒÀ•Œ). Fig. 5. Sedimentation equilibrium of purified Coryne bacterium sarcosine oxidase by the meniscus depletion Gel a was stained with 0.1% Coomassie Brilliant Blue method. The run was carried out at 12,919 rpm and R-250. Gel b was photographed under ultraviolet 20•Ž with 0.036% enzyme solution in 0.01 m potassium illumination (360 nm) in 7% acetic acid. phosphate buffer containing 0.1 M KCl, pH 8.0. r: radial distance. f: fringe displacement.

method of Yphantis (8). A linear relationship was obtained when the logarithm of fringe dis placement, Inf., was plotted against the square of radial distance, r2; this supports the homogeneity of the enzyme preparation (Fig. 5). Absorption spectra and identification of FAD:

The absorption spectrum of the purified enzyme

displayed maxima at 276, 369, and 451, being Fig. 3. Ultracentrifugal schlieren diagram of purified characteristic of a flavoprotein. The extinction Corynebacterium sarcosine oxidase. Ultracentrifugation coefficient of the enzyme at 280 nm (E1%1cm) and the sedimentation was carried out at 55,047 rpm at 20•Ž. The enzyme concentration was 0.728% in 0.01 M ratio of E280nm/E451nm were 13.1 and 12.1, respec

potassium phosphate buffer containing 0.1 M KCl, pH tively (Fig. 6). For analysis of the bound flavin, 8.0. The photograph was taken at 40 min after reach the enzyme (50.5 nmol) was treated at 100•Ž for ing full speed, with a bar angle of 70?. 5 min, and the precipitate was collected by cen

trifugation. The resulting precipitate was dissolved

at 20•Ž was calculated to be 9.35S by extrapolating in 1 ml of Buffer A and digested with 5 mg of

the protein concentration to zero (Fig. 4). The pronase at 37•Ž for 15 h. After heat-inactivation molecular weight of sarcosine oxidase was esti and centrifugation, the solution was treated with

mated to be 174,000 by the meniscus depletion 100 ƒÊg of phosphodiesterase at 37•Ž for 30 min.

Vol. 89, No. 2, 1981 604 M . SUZUKI

TABLE ‡U. Identification of the liberated flavin and covalently bound flavin on TCA-treated sarcosine oxidase. The purified enzyme (33 mg) was dissolved in 1 ml of Buffer A and treated with 1 ml of 10% (w/v) TCA at 100•Ž for 5 min. The suspension was cooled and centrifuged at 15,000 •~ g for 15 min. The super natant was lyophilized, dissolved in 0.2 ml of distilled water, and analyzed for the identification of flavin by thin layer chromatography (cellulose). The precipitate was collected by centrifugation as above and washed with 1% (w/v) TCA. The precipitate was suspended in 7 ml of 0.3 M potassium phosphate (pH 7.7) and 0.1 mg

Fig. 6. Absorption spectrum of purified Corynebac each of trypsin and chymotrypsin/mg protein were teriwn sarcosine oxidase. Spectra were recorded with added. After incubation at 37•Ž for 4 h, the incubation 0.045% sarcosine oxidase in 0.01 M potassium phosphate mixture was heated at 100•Ž for 5 min and filtered by buffer, pH 8.0, in the ultraviolet region and with 0.33% means of a Millipore membrane. The flavin peptide solution in the visible region. -, native enzyme; solution containing 7.8 •~ 10-5 mol as FAD in a volume native enzyme in the presence of 40 mM sarcosine; of 3 ml (pH 6.5) was treated with 0.05 unit of venom -•E- , native enzyme in the presence of 6 mM di phosphodiesterase at 37•Ž for 4 h. The pH of the flavin thiothreitol. peptide solutions was adjusted by the addition of 3 N HCl. (a): The liberated flavin (supernatant fraction). Solvent system 1; n-butyl alcohol-acetic acid-water The reaction was stopped and AMP was estimated =4 : 1 : 1. Solvent system 2; n-butyl alcohol-methyl enzymatically as described in " MATERIALS alcohol-5% Na2HPO4=4 : 1 :2. (b): The covalently

AND METHODS." The supernatant after heat bound flavin (precipitate fraction). Excitation wave length, 450 nm; Emission wavelength, 530 nm. treatment of sarcosine oxidase was digested and AMP was measured in the same way as above.

Approximately 42.5 nmol of AMP was recovered

in the supernatant and 44.5 nmol of AMP was found in the precipitate fraction. Table II-a shows

that the non-covalently bound flavin, obtained by 10% TCA extraction of the purified enzyme, was identified as FAD by thin layer chromatography.

The covalently bound flavin-peptide was prepared

by proteolytic digestion of the 10% TCA-precipi

tate. The fluorescence at pH 3.0 increased on nucleotide phosphodiesterase treatment, suggesting

that the flavin is at the dinucleotide level (Table ‡U-b). Therefore, this enzyme contains 1 mol of

non-covalently bound FAD and 1 mol of cova

lently bound FAD per mol of enzyme. Stoichiometry of the enzyme reaction: Hydro

gen peroxide formed from sarcosine was stoichi ometrically converted to formaldehyde by means of the catalase-methyl alcohol system (10). Table

‡V shows that the quantities of sarcosine and oxygen consumed in the reaction mixture each dimethylurea, 1-methylguanidine, 1-methylhydan coincided with that of glycine, formaldehyde or toin, sarcosyl-glycine, and sarcosyl-sarcosine were hydrogen peroxide formed during the reaction. examined as possible substrates for sarcosine Substrate specificity: L-Amino acids, D-amino oxidase. The enzyme activity for each substrate acids, N-methyl-L-amino acids, N-ethyl-glycine, was determined in terms of formaldehyde forma N,N-dimethylglycine, glycine, betaine, choline, 1,3 tion, hydrogen peroxide formation, or oxygen

J. Biochem. BACTERIAL SARCOSINE OXIDASE 605

TABLE ‡V. Stoichiometry of the sarcosine oxidase reaction,

a The reaction mixture contained 20 ƒÊmol of sarcosine and 300 ƒÊmol of glycylglycine buffer, pH 8.3, in a volume

of 4.9 ml. The tip of the sensor of the oxygen analyzer was put into the mixture and its surface was covered with liquid paraffin. After preincubation at 37•Ž for 5 min, 0.1 ml of sarcosine oxidase (8.4 U/ml) was injected into the reaction mixture. b The reaction mixture contained the same components as above together with 10,000 units of catalase and 0.3 ml of 99.5% methyl alcohol. The decrease in the amount of dissolved oxygen was measured for 10 min with an oxygen analyzer. At the same time, 0.5 ml of the mixture was taken out and mixed with an equal volume of 0.5 N acetic acid. Sarcosine, glycine and formaldehyde were estimated as described in the text. The value of H2O2 in Table III is given as half the amount of HCHO formed in the reaction mixture (2).

TABLE ‡W. Km and kcat of Corynebacterium sarcosine oxidase. Substrate concentrations were changed between 2.5 and 50 mM. Assay A: The enzyme activity was determined by measuring the amount of formaldehyde as described in " MATERIALS AND METHODS." Assay B: The enzyme activity was determined by measuring the amount of hydrogen peroxide formed, with an assay mixture (0.5 nil) containing 0.08 unit of sarcosine oxidase, 1.25-25 ƒÊmol of sarcosine, 30 ƒÊmol of potassium phosphate buffer (pH 7.0), 0.6 ƒÊmol of dimethylaniline, 0.05 ƒÊmol of 4-aminoantipyrine, and 0.54 unit of peroxidase. The reaction mixture was incubated at 37•Ž for 10 min. The reaction was stopped with 3 ml of 0.5 N acetic acid and the absorbance of the solution was measured at 550 nm.

consumption, as described in " MATERIALS TABLE V. Hydrogen-acceptor specificity of Coryne bacterium sarcosine oxidase. The reaction mixture con AND METHODS." tained 3 ml of 0.2 M sarcosine, 0.02 ml of dilute enzyme, The enzyme showed no activity towards these 1 ml of 0.3 M potassium phosphate buffer (pH 7.7) and compounds except for sarcosine, N-methyl-L 1 ml of each acceptor at various concentrations. - alanine and N-ethyl-glycine. The Km and kcat

(Vmax/E0) values of the enzyme for sarcosine, N-ethyl-glycine and N-methyl-L-alanine were deter mined from Lineweaver-Burk plots (Table ‡W). Hydrogen-acceptor specificity: The results of this experiment (given in Table V) show that sarcosine oxidase is very specific for oxygen as an acceptor. Of the other acceptors tried, PMS, 2,6-DCPI and ferricyanide served as hydrogen acceptors, but their Vmax/Km values were much smaller than that for 02. Methylene blue showed no detectable reaction; the behavior in this case

Vol. 89, No. 2, 1981 606 M. SUZUKI

was in marked contrast with that of sarcosine dehydrogenase of Pseudomonas putida (3). Effects of pH and temperature on activity and

stability: The optimal pH of the enzyme activity

was 7.7 in potassium phosphate buffer and 8.0

8.5 in glycylglycine buffer (Fig. 7). When the

enzyme solutions were assayed at pH 8.0 after standing for 24 h at 5•Ž and at various pHs, the

enzyme was found to be stable in the pH range between 7 and 10. The enzyme exhibited the

highest activity at 37•Ž in glycylglycine buffer Fig. 7. Effect of pH on sarcosine oxidase activity. The reaction mixture contained 60 ƒÊmmol of sarcosine, (pH 8.3) but its activity was lost completely on heating at 45•Ž for 10 min. 0.014 unit of sarcosine oxidase and 30 ƒÊmol of the in dicated buffer at various pH values. The assay was Effects of metal ions and chemicals on activity: carried out at 37•Ž for 10 min. •›, potassium phos As shown in Table VI, the enzyme activity was

phate; •œ, glycylglycine-NaOH; A, Tris-HCl. inhibited by Zn2+, Cu2+, Cd2+, Hg2+, Ag+, p-CMB, iodoacetamide, N-ethylmaleimide, CN-, o-phenan

throline, and sodium lauryl sulfate. There was

TABLE ‡Y. Effects of metal ions and some inhibitors no inhibitory effect with metal ions and chemicals on Corynebacterium sarcosine oxidase activity. The such as Cat+, Mg2+, Fe3+, ƒ¿,ƒ¿•Œ-dipyridyl, EDTA, reaction mixture contained (0.5 ml) 0.015 unit of sarco 2-mercaptoethanol, glutathione (reduced form), sine oxidase, 30 ƒÊmol of glycylglycine buffer, pH 8.3, dithiothreitol, NaN3, NaF, and NaAsO2. Acetate, and the indicated amounts of metals or inhibitors. After propionate, formaldehyde, and acetaldehyde in preincubation for 5 min at 37•Ž, the reaction was start hibited the enzyme activity by about 50% at the ed by the addition of 60 ƒÊmol of sarcosine. Formal concentration of 0.02 M. dehyde formed was measured by the method described in the text. DISCUSSION

From the stoichiometry data it could be concluded that sarcosine oxidase from Corynebacterium sp. U-96 catalyzes the following reaction.

No enzymatic activity was observed with N methyl-L-amino acids other than sarcosine and N-methyl-L-alanine. In this respect Corynebac terium sarcosine oxidase differs from N-methyl - L-amino acid oxidase [EC 1.5.3.21 of rat liver, which oxidizes many kinds of N-methyl-L-amino acids (11). Sarcosine oxidase from Corynebacterium sp. U-96 had a molecular weight of 174,000, which is very close to that of sarcosine dehydrogenase of Pseudomonas (170,000), but different from that of sarcosine oxidase purified from Cylindrocarpon didymum (45,000) grown in a choline medium (12). Pseudomonas sarcosine dehydrogenase consists of four identical subunits (3). In contrast, Coryne bacterium sarcosine oxidase contained four non

J. Biochem. BACTERIAL SARCOSINE OXIDASE 607 identical subunits termed A, B, C, and D. The The author is very grateful to Prof. T. Minoda, Uni molecular weights estimated by SDS disc gel versity of Tokyo, Japan, for his guidance and also to electrophoresis were 110,000, 44,000, 21,000, and Dr. N. Saito for valuable suggestions and discussions. 10,000 for subunits A, B, C, and D, respectively. Thanks are also due to Drs. M. Mogi, F. Yoshida, The sum of these values is close to the molecular D. Fukushima and M. Nagasawa for their support weight of the native enzyme. To ensure complete and encouragement, and to Drs. I. Koshiyama and M. Kikuchi for their help in ultracentrifugal analysis and denaturation of the protein, the enzyme was treated kind suggestions. The technical assistance of Miss T. with 6 to guanidine hydrochloride, then alkylated Kurokawa is also gratefully acknowledged. and subjected to SDS disc gel electrophoresis. The same results were obtained when the alkylated REFERENCES enzyme was used. Fluorescence derived from flavin covalently bound to the enzyme was found 1. Frisell, W.R. & Mackenzie, C.G. (1970) in Methods only in subunit B. The molar ratio of A : B : C : in Enzymology (Tabor, H. & Tabor, C., eds.) Vol. 17, D was calculated to be 1.02 :1.00 : 0.94 :1.03 by Part A, pp. 976-981, Academic Press, New York 2. Hoskins, D.D. & Bjur, R.A. (1964) J. Biol. Chem. densitometric scanning of the stained gel. These 239,1856-1863 results show that the enzyme is composed of four 3. Oka, I., Yoshimoto, T., Rikitake, K., Ogushi, S., & different kinds of subunits. Tsuru, D. (1979) Agric. Biol. Chem. 43, 1197-1203 Thus, sarcosine oxidase from Corynebacterium 4. Buchanan, R.E. & Gibbons, N.E. (1974) Bergey's might be a new fiavoprotein with two kinds of Manual of Determinative Bacteriology 8th Ed., pp. FAD. Calculation showed this enzyme contains 599-632, The Williams & Wilkins Co., Baltimore I mol of covalently bound FAD and 1 mol of 5. Nash, T. (1953) Biochem. J. 55, 416-421 non-covalently bound FAD per mol of enzyme. 6. Jaworek, D., Gruber, W., & Bergmeyer, H.U. In this respect, sarcosine oxidase of Corynebac (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.) Vol.4, pp. 2127-2131, Academic Press, terium differs from that of C. didymum, which New York contains I mol of covalently bound FAD per mol 7. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404 of enzyme (12), and also differs from other fiavo 427 protein oxidases such as glucose oxidase from 8. Yphantis, D.A. (1964) Biochemistry 3, 297-317 Aspergillus niger (13), L-amino acid oxidase from 9. Weber, K., Pringle, J.R., & Osborn, M. (1972) Crotalus adamanteus (14) and tyramine oxidase Methods in Enzymology (Hirs, C.H.W. & Timasheff, from Sarcina lutea (15), which have 2 mol of S.N., eds.) Vol. 26, Part C, pp. 3-27, Academic non-covalently bound FAD per mol of enzyme, Press, New York or choline oxidase of C. didymum (16), which has 10. Kageyama, N. (1971) Clinica. Chimica. Acta 31, 421-426 2 mol of covalently bound FAD per mol of en 11. Hirohata, R., Moriya, M., Izumi, N., Kenmochi, K., zyme. The functions of the two types of FAD Ota, N., & Fujii, S. (1951) Koso Kagaku Sympo. in the enzyme catalysis, and the physico-chemical (in Japanese) 6, 46 characteristics of the enzyme are currently under 12. Mori, N., Sano, M., Tani, Y., & Yamada, H. (1980) investigation. Agric. Biol. Chem. 44, 1391-1397 Incidentally, this enzyme, sarcosine oxidase, 13. Swoboda, B.E.P. & Massey, V. (1965) J. Biol. Chem. can be used for the enzymatic determination of 240,2209-2215 serum creatinine and creatine by coupling it with 14. Wellner, D. & Meister, A. (1960) J. Biol. Chem. bacterial creatinine amidohydrolase and creatine 235,2013-2018 amidinohydrolase. This is useful in the diagnosis 15. Kumagai, H., Matsui, H., Ogata, K., & Yamada, H. (1969) Biochim. Biophys. Acta 171, 1-6 of kidney trouble and muscular dystrophy (17). 16. Tani, Y., Mori, N., Ogata, K., & Yamada, H. (1979) Agric. Biol. Chem. 43, 815-820 17. Suzuki, M. & Yoshida, M. (1979) Rinsho Kagaku Sympo. (in Japanese) 19, 196

Vol. 89, No. 2, 1981