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[220] d -Amino acid oxidase and its complexes (hog kidney)
608
AMINO ACID OXIDATION
[2201
Serine, phenylalanine, and tryptophan have quite high nonenzymatic rates. In the case of the latter, lowering the pyridoxal-P concentration to 1.5 × 10-4 M or less decreases the nonenzymatic reaction greatly with respect to that with peroxidase. 4 The increment ofdecarboxylation due to added enzyme is greatest with methionine as substrate, followed in order by serine, methionine sulfoxide, phenylalanine, alanine and tryptophan. Results obtained by Hill and Mann s using a similar system but containing p-cresol show that glycine, homoserine, aspartic acid, glutamic acid, ornithine, lysine, and asparagine are also decarboxylated. Kinetic Properties. The Km for DL-methionine is 7.8 × 10-4 M. Activity of Peroxidases from Other Sources. Enzyme preparations that have similar properties to the horseradish peroxidase system have been obtained from cabbage leaves, turnip root, squash fruits, and mustard leaves. An extract obtained by digitonin treatment of cell particulates is treated with (NH4)2SO4 to yield the enzyme fraction having the decarboxylase activity. The mustard and cabbage extracts when tested for peroxidase are highly active. A partially purified lactoperoxidase has also catalyzed the decarboxylation of methionine. 4V. M. Riddle and M. Mazelis, Plant Physiol., 40, 481 (1965). ~J. M. Hill and P.J.G. Mann, Biochem.J., 99, 454 (1966).
[220]
D-Amino Acid Oxidase I and Its Complexes (Hog Kidney) By KUNIO YAGI R CH C O O H + O2 + H~O
I
NH2
) R C C O O H + NHa + H2Oz
II
O
The preparation method reported by Negelein and Br6mel la'2 is based on isolation of the apoenzyme of this oxidase. Even though the preparation obtained by this classical method afforded fundamental knowledge about this enzyme, the sample itself was impure and unstable. Nowadays, this method has been almost completely replaced by
1EC 1.4.3.3; D-Amino acid:oxygen oxidoreductase (deaminating). laE. Negelein and H. Br6mel, Biochem. Z., 300, 925 (1939). 2K. Burton, Vol. II, p. 199.
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D-AMINO ACID OXIDASE AND ITS COMPLEXES
609
n e w e r m e t h o d s 3-6 c h a r a c t e r i z e d by the use o f a stabilizer. Since the e n z y m e was f o u n d to f o r m a stable c o m p l e x with benzoate, z-'° it is most easily isolated in the f o r m o f an e n z y m e - b e n z o a t e c o m p l e x . T h e enz y m e - b o u n d b e n z o a t e can be easily e x c h a n g e d for the substrate; this c o m p l e x is a suitable starting material for the p r e p a r a t i o n o f free h o l o e n z y m e a n d its a p o e n z y m e . Since this c o m p l e x can be r e g a r d e d as an e n z y m e - s u b s t r a t e c o m p l e x model, al it should be strictly distinguished f r o m free h o l o e n z y m e . T h e reaction s e q u e n c e o f the a n a e r o b i c catalysis o f this e n z y m e is e x p r e s s e d as E + SH2 ~- E --SH2 ~- purple intermediate ~- EH.TS ~ EH~ + S w h e r e SH2 r e p r e s e n t s the substrate, a n d S the p r o d u c t . Since the p u r p l e i n t e r m e d i a t e c o m p l e x , '2 w h e n it accumulates, is s p o n t a n e o u s l y conv e r t e d into the s e m i q u i n o i d e n z y m e a n d the substrate radical, 13 the p r e p a r a t i o n o f the s e m i q u i n o i d e n z y m e is also described h e r e in addition to those o f the p u r p l e i n t e r m e d i a t e a n d the fully r e d u c e d forms, a l t h o u g h the s e m i q u i n o i d e n z y m e is not involved in the enzymatic reaction sequence.
Assay Method Principle. T h e e n z y m e activity has b e e n assayed by m e a s u r i n g , m a n o m e t r i c a l l y 2 or polarographically, 6,14 the rate o f o x y g e n cons u m p t i o n d u e to the oxidation o f substrate by this e n z y m e . Recently, a new assay m e t h o d was r e p o r t e d '5 in which the a m o u n t o f a m m o n i a liberated t h r o u g h the oxidative d e a m i n a t i o n o f substrate by this enz y m e is m e a s u r e d directly by the i n d o p h e n o l m e t h o d . Also, a m e t h o d based on the inactivation o f catalase by 3-amino-l,2,4-triazole in the
3F. P. Veitch and R. McComb,J. Am. Chem. Soc., 78, 1363 (1956). 4H. Kubo, T. Yamano, M. Iwatsubo, H. Watari, T. Soyama, J. Shiraishi, S. Sawada, N. Kawashima, S. Mitani, and K. Ito, Bull. Soc. Chim. Biol., 40, 431 (1958). 5V. Massey, G. Palmer, and R. Bennett, Biochim. Biophys. Acta, 48, I ( 196 t ). 6K. Yagi, M. Naoi, M. Harada, K. Okamura, H. Hidaka, T. Ozawa, and A. Kntaki, J. Biochem., 61,580 (1967). 7K. Yagi, T. Ozawa, and M. Harada, Nature, 184, 1938 (1959). SK. Yagi, T. Ozawa, and M. Harada, Nature, 188, 745 (1960). 9K. Yagi and T. Ozawa, Biochim. Biophys. Acto, 56, 413 (1962). '°K. Yagi and T. Ozawa, Biochim. Biophys. Acta, 56, 420 (1962). 11K.Yagi, Advances in Enz';molog3,, 27, 1 (1965). '2In aerobic catalysis, this intermediate reacts with oxygen. '3K. Yagi, K. Okamura, M. Naoi, N. Sugiura, and A. Kotaki, Biochim. Biophys. Acta. 146, 77 (1967). '*W. Kielley, Vol. VI, p. 272. 'ST. N agatsu and K. Yagi,J. Biochem., 60, 219 (1966).
610
AMINO ACID OXIDATION
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p r e s e n c e o f H202 g e n e r a t e d by the a m i n o acid oxidase was r e p o r t e d for the detection o f a small a m o u n t o f oxidase activity. TM Usually, however, the p o l a r o g r a p h i c m e t h o d is r e c o m m e n d e d because o f its simplicity and rapidity. Procedure. T o 10 ml o f a m i x t u r e o f 0.1 M D-alanine 17 and 1.0 X 10 -5 M FAD in 0.1 M sodium p y r o p h o s p h a t e buffer, p H 8.3, 0.03-0.05 ml o f the e n z y m e solution (containing about 2 0 - 3 0 / x g o f e n z y m e protein) is a d d e d with constant mechanical stirring. T h e o x y g e n c o n s u m p t i o n is m e a s u r e d at 20 ° with a p o l a r o g r a p h i c o x y g e n electrode (e.g., Beckman O x y g e n Sensor) c o n n e c t e d to a r e c o r d e r . T h e e x p e r i m e n t a l arrangem e n t described by Kielley 14 is r e c o m m e n d e d . Enzymatic Activity and Specific Activity. O n e unit o f enzymatic activity is d e f i n e d as the a m o u n t that catalyzes the reaction resulting in the cons u m p t i o n o f 1 m i c r o m o l e o f o x y g e n per m i n u t e u n d e r the assay conditions. T h e specific activity is calculated as the activity per milligram o f protein. Protein is d e t e r m i n e d by the biuret method. TM
Purification Procedure Using benzoate as a stabilizer d u r i n g the purification p r o c e d u r e , a p u r e crystalline e n z y m e - b e n z o a t e c o m p l e x can be obtained f r o m hog kidney. This p r o c e d u r e was first devised by Kubo et al., 4 d e v e l o p e d by Massey et al., 5 and f u r t h e r i m p r o v e d by Yagi et alp F r o m this complex, h o l o e n z y m e and a p o e n z y m e can be p r e p a r e d . Purification can be carried out at r o o m t e m p e r a t u r e unless otherwise stated.
Purification and Crystallization of the Benzoate Complex of D-Amino Acid Oxidase Step 1. T h e capsule and fat are r e m o v e d f r o m hog kidney and discarded. After slicing, the red medulla is r e m o v e d and discarded; 250-g quantities o f sliced cortex are h o m o g e n i z e d in 1 liter o f buffer A TM in a W a r i n g B l e n d o r at half speed for the initial 30 seconds and at full speed for 2.5 minutes. F r o m 1 kg o f the cortex, 4.65 liters o f the h o m o g enate are obtained. T h e h o m o g e n a t e is adjusted to p H 7.6 by a d d i n g 1 N N a O H ; it is tLen slowly heated to 40 ° in a water bath set at 45 ° and 1ell. Scannone, D. Wellner, and A. Novogrodsky, Biochemistry,3, 1742 (1964). 17DL-Alanine can be used, because L-alanine does not affect the activity of this enzyme at this concentration. lSA. G. Gornall, C.J. Bardawill, and M. M. David, J. Biol. Chem., 177, 751 (1949); see also Vol. III, p. 450. ~aBufferA: 1.67 × 10-2 M sodium pyrophosphate buffer, pH 8.3, containing 0. 1% sodium benzoate.
[220]
D-AMINO ACID OXIDASE AND ITS COMPLEXES
611
maintained at 40 ° for 10 minutes. T h e n the h o m o g e n a t e is b r o u g h t to pH 5.2 by a d d i n g 1 N acetic acid, heated again to 40 °, and mainrained at 40 ° for 5 minutes. By centrifugation at 2200 g [br 10 minutes, a brownish red supernatant (ca. 3.6 liters) is separated from the pellet. Step 2. A m m o n i u m sulfate (176 g per liter) is a d d e d to the supernatant, and the mixture is allowed to stand for 1 h o u r at 5°. By centrifugation at 3000 g for 20 minutes, the precipitate is separated and dissolved in 100 ml of buffer A. T h e pH of this yellowish red solution is adjusted to 5.2 by the addition of 1 N acetic acid. T h e solution is heated to 55 ° in a water bath set at 60 °, maintained at 55 ° for 10 minutes, and is immediately centrifuged at 15,/)00 g for 10 minutes. A reddish yellow supernatant (ca. 115 ml) is obtained (first heat treatment). A m m o n i u m sulfate (114 g per liter) is a d d e d to the supernatant; after standing t o t 1 h o u r at 5°, the mixture is centrifuged at 15,000 g for 10 minutes and the s u p e r n a t a n t is discarded. T h e precipitate is dissolved in 50 ml of" buffer A. T h e solution is adjusted to pH 5.2 by adding 1 N acetic acid and then heated to 58 ° in a water bath set at 63 ° and mainrained at 58 ° for 3 minutes. After centrifugation o f the sample at 15,000 g for 10 minutes, the precipitate o f d e n a t u r e d protein is discarded. An orange yellow supernatant (ca. 50 ml) is obtained. By this second heat treatment, "° the concomitant catalase activity is usually eliminated. Step 3. A m m o n i u m sulfate (114 g per liter) is a d d e d to the supernatant, and after standing for 1 h o u r at 5°, the mixture is centrifuged at 15,000 g-for 10 minutes. T h e precipitate is dissolved in a minimal volume (ca. 10 ml) of buffer A. T h e enzyme solution is applied to a column of calcium phosphate gel-cellulose 't (2 x 15 cm) equilibrated previously with 0.05 M sodium phosphate buffer (pH 5.1). T h e enzyme is eluted with the same buffer; the enzyme moves down as a yellow band and is separated from brownish red impurities that remain at the upper part of the c o l u m n ) T h e eluate is r e c h r o m a t o g r a p h e d by the same procedure. T h e yield is 135 m g (see Table I). Step 4. T h e r e c h r o m a t o g r a p h e d eluate is b r o u g h t to 20% saturation with a m m o n i u m sulfate; after standing for 1 h o u r at 5°, the precipitated enzyme is separated by centrifuging at 15,000 g tot" 10 minutes and is dissolved in a minimal volume of buffer A. This solution is chilled in an
z°When a rapid preparation is n e e d e d , one can substitute h)r this step, a single heating (60 ° for 3 minutes), although the purity o f the p r e p a r a t i o n at this stage is somewhat lowered. alCalcium phosphate gel-cellulose: a mixture of calcium p h o s p h a t e gel =2 and cellulose p o w d e r ( 1: 15-30 w/w). 2'-'See Vol. 1, p. 98.
612
[220]
AMINO ACID OXIDATION
ice b a t h a n d a f e w d r o p s o f s a t u r a t e d a m m o n i u m
sulfate solution are
a d d e d to m a k e t h e s o l u t i o n slightly t u r b i d . A f t e r s t a n d i n g f o r s e v e r a l d a y s at 5 ° , t h e y e l l o w n e e d l e crystals w h i c h a p p e a r e d a r e c e n t r i f u g e d at 10,000 g f o r 10 m i n u t e s a n d d i s s o l v e d in b u f f e r A (ca. 7 m g / m l ) . T h e s o l u t i o n o f t h e c r y s t a l l i n e e n z y m e t h u s o b t a i n e d h as a b s o r p t i o n p e a k s at 3 7 9 m t t a n d 4 6 3 m/z a n d a m a r k e d s h o u l d e r at 4 8 6 m t t as s h o w n by c u r v e / i n Fig. 1A.
2.0
-~A
J~3
4I 300
B
400
III
500
3zI
I
0
400
500 Wovelength (mp.)
600
FiG. 1. Absorption and circular dichroic spectra of D-amino acid oxidase and its complexes. (A) Absorption spectra. (B) Circular dichroic spectra. Curves: 1, benzoate complex (the enzyme was mixed with 1 x 10-a M benzoate); 11, holoenzyme; 1H, purple intermediate (the enzyme was mixed with 5 x 10-2 M D-alanine in the presence of 1 × 10-j M lithium pyruvate and 5 × 10-z M ammonium sulfate); IV, semiquinoid enzyme complex with products (the enzyme was half-reduced with dithionite and was mixed with 3 x 10-2 M lithium pyruvate and 1 x 10-2 ~1 ammonium sulfate)'. V, fully reduced enzyme (the enzyme was reduced with 5 x 10-2 M D-alanine); VI, enzyme complex with Al-piperidine 2-carboxylate (the enzyme was mixed with 1 x 10-3 M this compound). The enzyme concentration was fixed at 1.2 × 10-4 M with respect to FAD. Measurements were made at room temperature. Unit of [0]: deg.cm2/dM.
Typical data for the overall purification procedure of the benzoate c o m p l e x a r e s u m m a r i z e d in T a b l e I.
[220]
D-AMINO
613
ACID OXIDASE AND ITS COMPLEXES
TABLE I SUMMARY OF PURIFICATION PROCEDURE OF BENZOATE COMPLEX OF D-AMINO ACID OXIDASE
Total volume (ml) Summary of procedure Step 1: Homogenate 4640 Crude extract 3640 Step 2: lst heat treatment 115 2nd heat treatment 52 Step 3: 1st column eluate 24 2nd column eluate 6.8 Step 5: Sephadex eluate .
Total activity (units)" Total KCN protein -- 10 n ~ l (mg) 860 740 710 11211 1150 930 . .
1720 1470 1420 1120 1150 930 .
182,000 26,000 1,370 760 200 135
Specific activity Recovery Degree (units/mg) of of KCN enzyme purifi-10 mM (%) cation 0.0047 0.028 0.52 1.47 5.8 6.9 8.8
0.0095 0.057 1.04 1.47 5.8 6.9 8.8
100 86 83 65 67 54 -
l 6 110 156 620 730 930
"A unit of activity is defined as the number of micromoles of oxygen consumed per minute.
The preparation obtained in step 4 is still too crude for physicochemical characterization; the following procedures are included for its further purification/a'24 Step 5. About 500 mg of the crystals of the benzoate complex obtained (usually from 10 kg of hog kidney slices) in step 4 is dissolved in 30 ml of buffer A and the solution is dialyzed against 3 liters of the same buffer for 24 hours at 5 °. The dialyzed solution is applied to a column of" Sephadex G-75 (2.5 x 35 cm), equilibrated previously with buffer A. The loading ratio is 10-20 mg of protein per gram of Sephadex. The enzyme is eluted with the same buffer, and yellow fractions having a ratio of about 1 mole of FAD per 50,000 g of protein are collected. Step 6. Crystallization of the benzoate complex fl'om the eluate obtained in step 5 is made in the same manner as described in step 4. Fine needle crystals are usually obtained, and after the preparation '-':~Further purification is also possible by repeating the crystallization 2-'~; times, though the yield becomes faMy low. '-'4As a simpliiied preparation of purified benzoate complex solution, the following procedure is recommended. 2~ The eluate from calcium phosphate gel-cellulose column is heated a~ 60 ° for 3 minutes at pH 5.2 and centrifuged at 15,000 g for 10 minutes. To the supernatant ammonium sulfate (I 1.4 g/100 ml) is added, and the mixture is centrifuged at 15,000 g for 10 minutes. The precipitate is dissolved in buffet" A (ca. 20 mg/ml). The solution containing about 500 mg of benzoate complex is applied to a column o! Sephadex (;-75 (5 × 40 crn) previously equilibrated with 0.01% aqueous solution of sodium benzoate and eluted with the same solution. Fractions having a ratio below 50,000 g of protein pet" mole of FAD are collected. The recovery is above 60% with respect to FAD. 2aK. Yagi, N. Sugiura, H. ()hama, and A. Kotaki,J. Biochem., 68,572 (1970).
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AMINO ACID OXIDATION
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has stood for several days, large hexagonal crystals appear in the solution. The protein moiety of this crystalline benzoate complex obtained from the Sephadex gel eluates is chemically homogeneous as j u d g e d by the N-terminal analysis. The crystals are a 1:1 complex composed of the oxidized enzyme (on the molecular basis of 50,000) and benzoateY 6
Preparation and Crystallization of the Holoenzyme 6 The holoenzyme is prepared from the crystalline benzoate complex by repeated expulsion of the benzoate from the complex with an excess of D-alanine. It is preferable to carry out this procedure in an ice bath. About 500 mg of the crystalline benzoate complex is dissolved in 50 ml of buffer B 27 (enzyme concentration:2 × 10 -4 M with respect to FAD) and mixed with 2 g of DL-alanine (final concentration 2.2 × 10 -1 M D-alanine); 6 g of solid ammonium sulfate is added to this solution (ca. 20% saturation). The pH of the mixture is adjusted to 6.1 by adding 1 N acetic acid. The purple precipitate formed is collected immediately by centrifuging at 15,000 g for 10 minutes. This treatment is repeated 3 times. The purple precipitate is dissolved in a small volume of buffer B (ca. 15 ml), and the solution is dialyzed overnight against 2 liters of the same buffer at 5 ° with mechanical stirring. The dialyzing medium is changed 2 times. A transparent deep yellow solution of the oxidized holoenzyme is obtained. To j u d g e the complete removal of benzoate, a splitting in the absorption peak at around 375 m/z is a good indication (see curve H in Fig. 1A, and also Absorption Spectra under Properties). To crystallize the holoenzyme, ammonium sulfate is added to the above-mentioned dialyzed preparation (enzyme concentration: ca. 6 × 10 -4 M with respect to FAD) to 1% saturation at 25 °. Then the pH is adjusted to 8.3. The solution is stored at 5 ° for about 10 days, and yellow hexagonal crystals are obtained. 28
Preparation of the Apoenzyme of D-Amino Acid Oxidase For the cleavage of the holoenzyme into the apoenzyme and FAD, the classical method according to Negelein and Br6meP a'2 has been widely used, a method which is also applicable to both the holoenzyme and its benzoate complex. However, the yield of the active apoenzyme is very low because of considerable denaturation due to t h e acid treatment. ~K. Yagi and M. Harada,J. Biochem., 57,463 (1965). 27Buffer B: 1.67 x 10-2M sodium pyrophosphate buffer, pH 8.3. ~SThe crystal of the holoenzyme can also be obtained by dialyzing the purple intermediate crystal against buffer B at 5°. 29 ~gK. Yagi and T. Ozawa,J. Biochem., 54, 202 (1963).
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D-AMINO ACID OXIDASE AND ITS COMPLEXES
615
Recently, improved methods for the preparation of the apoenzyme, using column chromatography with hydroxylapatite 3° or with TEAEcellulose 6 as adsorbant, have been reported. A simpler method depends upon dialysis in the presence of a high concentration of KBr. a' Preparation of Apoenzyme by Dialysis. The benzoate-free holoenzyme (1 x 10 -4 M with respect to FAD) is dialyzed at 4 ° against a solution of 0.1 M sodium pyrophosphate, pH 8.5, containing 3 × 10-3 M EDTA and 1 M KBr. The volume inside the dialysis sac is 5 ml; the volume of the dialysis medium is 1 liter. The dialyzing medium is changed 3 times over a 2-day period until the yellow color of the holoenzyme disappears. The dialyzing medium is then changed to 0.1 M sodium pyrophosphate buffer, pH 8.5, and this dialysis is continued for 2 days to accomplish complete removal of KBr. The yield of the apoenzyme with respect to its enzymatic activity is nearly 100%. Preparation of Apoenz~me b~, Chromatography on TEAE-cellulose. Two milliliters of the holoenzyme solution in buffer B (protein concentration: 2 × 10 -4 M with respect to FAD) are applied to a TEAE-cellulose column (2.5 x 40 cm) equilibrated previously with 5 mM sodium phosphate buffer, pH 5.2. The loading ratio is about 1 mg of protein per gram of ion exchanger. The apoenzyme is eluted with the same buffer at 5° at a flow rate of 5 ml/30 minutes, and it appears in the effluent usually between fraction 10 and fraction 13 (each fraction volume, 5 ml). A yellow band of FAD is left at the upper part of the column.
Preparation of the Intermediate Complex and Its Related States
P'mple Intermediate Complex'a'3z,aa The purple complex, a reaction intermediate of D-amino acid oxidase, is crystallized from the mixture of the holoenzyme, the substrate, Dalanine, and the products, pyruvate and ammonia, under anaerobic conditions. The crystals consist of equimolar amounts of the enzyme (on the molecular basis of 50,000) and the substrate moiety which is readily converted into the products on aeration. Since concentration of the purple intermediate in the solution is governed by the concentrations of the substrate and the products, a4 a quantitative study should adopt definite concentrations of the enzyme, the substrate and the products, which are mixed under strictly anaerobic conditions. However, for the crystallization of the purple intermediate, the original method starting :"~Y. Miyake, K. Aki, S. Hashimoto, and T. Yamano, Bioehim. Biophys. Acta. 105, 86 (1965). a'V. Massey and B. Curti, ]. Biol. Chem., 241,3417 (1966). a2K. Yagi and T. Ozawa, Bioehim. Biophys. Acta, 60,200 (1962). :~:~K.Yagi and 'F. Ozawa, Biochim. Biophys. Acta, 81, 29 (1964). :~4K. Yagi, K. Okamura, N. Sugiura, and A. Kotaki, Biochim. Biophys. Acta, 159, 1 (1968).
616
AMINO ACID OXIDATION
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from the enzyme-benzoate complex is recommended, because of its simplicity. In this case, some pyruvate is produced from D-alanine during the preparation procedure, and this product, together with the excess amount of another product, ammonia, added in the form of ammonium sulfate, affects the equilibrium to maintain the amount of the purple intermediate. The amount of pyruvate produced is sometimes sufficient, but in most cases it is necessary to add a small amount of powdered lithium pyruvate to make the solution deep purple; this is an adequate condition for crystallizing the purple intermediate. Crystallization Starting [rom the Enzyme-Benzoate Complex.a2,aa Pure crystalline enzyme-benzoate complex (0.2-1.0 g) is dissolved in 50 ml of buffer B, and 1 g of D-alanine is added with stirring. The pH of the solution is brought to 6.1 by adding 1 N acetic acid, and 6 g of ammonium sulfate is added to the solution. After standing at 5 ° for 30 minutes in an icebox, the precipitate is collected by centrifugation at 15,000 g for 10 minutes and dissolved in 50 ml of buffer B (final pH 7.5-7.7). Then D-alanine (about 1 g) is carefully added with stirring to the brownish yellow solution thus obtained, until the color of the solution turns to purple. The pH of the solution is brought to 6.1, and 6 g of ammonium sulfate is added. The purple colored precipitate is collected by centrifugation at 15,000 g for 10 minutes and is dissolved in a minimal volume of buffer B, so that the enzyme concentration becomes about 1 x 10-3 M with respect to FAD. The pH of the purple solution thus becomes 7.2, and the solution becomes about 5% saturated with respect to ammonium sulfate. If the pH is not 7.2, it should be adjusted exactly to this figure by the addition of acid or alkali. Powdered lithium pyruvate is carefully added to this solution until the solution becomes deep purple. The solution is transferred to a Thunberg-type tube filled with argon gas. At this moment, double refraction of flow is usually observed. After the preparation has stood at 5 ° overnight, a crop of purple crystals appears. The shape of the crystal is a hexagonal prism with bipyramids, a~ To check the conditions for crystallization, the following method is convenient; a small amount of the solution (0.02 ml) is placed on a piece of glass (such as a microscope slide) and sealed with petrolatum and a cover glass? ~ When the conditions are suitable, crystals form gradually and can be observed with a microscope. Crystallization Starting from the Holoenzyme. To obtain the purple intermediate solution, usually 3 ml of the holoenzyme (1.0 x 10 -4 M with respect to FAD dissolved in buffer B) is placed in the main chamber ~SK.Yagiand T. Ozawa,J. Biochem.,53, 162 ( !963).
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D-AMINO
ACID OXIDASE
AND ITS COMPLEXES
617
o f a T h u n b e r g - t y p e cuvette, and D-alanine (5.0 X 10-=' M final concentration), lithium p y r u v a t e (1.0 x 10 -1 M final concentration), and a m m o n i u m sulfate (5.0 x 10 -2 M final concentration) are placed in the sidearm, v'~ T o eliminate oxygen, evacuation and flushing with p u r e argon ,gas are carried out, alternately, several times; the contents o f the sidearm are then a d d e d to the main c h a m b e r . A d e e p - p u r p l e solution (pH 7.0-7.2), having a s p e c t r u m shown by curve I11 in Fig. I A, is o b t a i n e d ? ~ If the c o n c e n t r a t i o n o f the e n z y m e is increased to ca. 5 x 10 -4 M and a m m o n i u m sulfate to 5 % saturation, crystals o f the p u r p l e i n t e r m e d i a t e can be obtained. Usually a longer time is r e q u i r e d fi)r the crystallization than for that in the case starting f r o m the enzymebenzoate complex. Semiqui,oid Form of D-Amino Acid Oxidase
A solution o f the semiquinoid e n z y m e can be f o r m e d r a t h e r easily by anaerobic t r e a t m e n t with sodium dithionite 37 or by strong light irradiation 3s'39 of the h o l o e n z y m e u n d e r anaerobic conditions. On the o t h e r hand, the p u r p l e intermediate is c o n v e r t e d slowly into the semiquinoid e n z y m e u p o n storage in the dark. ~3 This conversion is accelerated by illumination. F r o m this solution, the semiquinoid f o r m o f the e n z y m e can be crystallized. 4°'4~ T h r e e milliliters o f the p u r p l e complex solution (ca. 2 x 10-4 M with respect to FAD) in a T h u n b e r g - t u b e , are illuminated by a 30 W tungsten lamp (e.g., Mazda daylight lamp) at a distance o f 20 cm for 48 hours at 5 ° u n d e r anaerobic conditions. T h e solution gradually turns to reddish purple. After the absorption s p e c t r u m reaches curve IV in Fig. I A, p o w d e r e d a m m o n i u m sulfate is a d d e d anaerobically to the solution until the c o n c e n t r a t i o n is 0.2 M (ca. 5 % saturation) and its p H is adjusted to 6.8-7.0. W h e n this solution is stored at 5 ° for 2 days, reddish b r o w n crystals appear. T h e crystal is a h e x a g o n a l prism with bipyramids. In the crystalline p r e p a r a t i o n , this semiquinoid e n z y m e exists as its c o m p l e x with coexisting substrate, D-alanine, a n d / o r products, pyruvate and a m m o n i a , t h o u g h these c o m p l e x e s are not involved in the enzymatic reaction sequence. :~qn this case, the purple intermediate present reaches 90% of its maximal fi)rmation? 4 :~T'I'.Nakamura, S. Nakamura, and Y. Ogura,J. Biochem., 54, 512 ( 1963). aSH. Watari, K-J. Hwang, K. Ashida, and K. Kinoshita, Biochim. Biophys. Acta, 128, 256 (1966). agV. Masseyand G. Palmer, Biochemistry, 5, 3181 (1966). 4°K. Yagi and N. Sugiura,J. Biochem., 60, 738 (1966), ~ K. Yagi, N. Sugiura, K. Okamura, and A. Kotaki, Biochim. BiophU. Acta, 151,343 (1968).
618
AMINO ACID OXIDATION
[220]
Crystallization of the Fully Reduced Form of D-Amino Acid Oxidase 34"42 The fully reduced form of this oxidase is produced by anaerobic reduction of the enzyme with excess D-alanine. Its crystallization can be performed by increasing the concentration of ammonium sulfate. The crystallization procedure is somewhat similar to that of the purple intermediate starting from the holoenzyme except for the absence of pyruvate. The key point of the preparation is, therefore, to carry out the procedure under strictly anaerobic conditions, which enables one to obtain the fully reduced form without significant contamination of pyruvate. To 6 ml of the enzyme solution containing 200 mg of the holoenzyme (6.7 x 10 -4 m with respect to FAD), 160 mg of powdered D-alanine (3 X 10-1 M final concentration) is added with gentle shaking under anaerobic conditions. The yellow color of the holoenzyme solution rapidly changes into purple and then gradually turns to a pale yellow, indicating that the enzyme is fully reduced. To this pale yellow solution, 168 mg of powdered ammonium sulfate (2.3 x 10 -1 M final concentration, ca. 5% saturation) is added with stirring and the resulting solution is stored at 5 ° under anaerobic conditions (gas phase, argon). After storing this solution over a week, a crop of pale yellow crystals appears. The shape of the crystals is a hexagonal prism, or a hexagonal prism with bipyramids. The crystals are a 1:1 complex composed of the fully reduced enzyme (on a molecular basis of 50,000) and the intact substrate, D-alanine.
Properties Absorption Spectra. The visible absorption spectrum of the holoenzyme is shown by curve H in Fig. 1A. The marked splitting of the peak at around 375 mft is characteristic. 43 When the enzyme combines with benzoate, the splitting of the absorption peak disappears with some hypochromism and a three-banded structure appears at an absorption peak around 460 m~ 43 (see curve I in Fig. 1A). The molar extinction coefficients for the holoenzyme and the benzoate complex at their absorption peaks, 455 m/.t and 463 m/~, respectively, are approximately 1.13 X 104. The extinction coefficient (E2s0) 1~ of the apoenzyme is 15.12 The purple intermediate complex is characterized by its broad absorption band in the vicinity of 550 mft (curve III), 13 whereas the semiquinoid enzyme is characterized by its absorption peak at 492 m/~ ( c u r v e IV). 3"&39"4°'41 On the other hand, the fully reduced form of the enzyme has no absorption peak in the visible wavelength region (curve 42K. Yagi and K. Okamura, Biochem. Biophys. Res. Commun., 21,399 (1965). 43A. Kotaki, M. Naoi, and K. Yagi,J. Biochem., 59, 625 (1966).
[220]
D-AMINO ACID OXIDASE AND ITS COMPLEXES
619
V). 34'42 Curve VI shows the spectrum of the complex of the oxidized enzyme with Al-piperidine 2-carboxylate, 44 which is formed from spontaneous cyclization of a-oxo-E-aminocaproate produced from Dlysine by this enzyme. 45 This spectrum is also characterized by a broad absorption band at the longer wavelength region, which is considered to be a charge transfer band. 44"46 A similar charge transfer band can also be observed by mixing the enzyme with other n-donors, such as 0-aminobenzoate, pyrroline 2-carboxylate, and indole 2-carboxylate. 44 Substrate Spec!ficitv. Although the substrate commonly used for study is D-alanine, in which case the molecular activity 6 (with respect to the molar concentration of FAD, measured by routine polarographic procedure at 20 °) is 435, the enzyme oxidizes various D-a-amino acids 47 and N-monoalkylated D-a-amino acids? 8 However, acidic D-a-amino acids are not oxidized. 49 Enzymatic oxidation of D-lysine is interrupted immediately by Al-piperidine 2-carboxylate, a strong inhibitor, [brmed from the reaction product through spontaneous cyclization. 4"~Glycine, ~° o-a-hydroxy acids (e.g., D-lactic acid), 51 and L-proline ~2,'~a are reported to be attacked by this enzyme at a slower rate. Electro~ Acceptors. Methylene blue and 2,6-dichlorophenolindophenol can serve as hydrogen acceptors, though they are not as active as molecular oxygen. 49 Inhibitors. Systematic investigation of the mechanism of inhibition using benzene derivatives 54 revealed that 0-COOH, ¢-NH2, and O-OH functions are responsible for competition with the substrate, competition with FAD, and combination with FAD, respectively. In accord with this indication, various carboxylic acids, including fatty acids, '~'~ some L-amino acids, 4"~,56benzoic acid, 57 and its derivatives, 54,.58and heterocyclic carboxylic acids (e.g., pyrrole 2-carboxylic acidSa), inhibit enzymatic 44V. Masse), and H. (;anther, Biockemist~)., 4, 1161 (1965). 4'5K. Yagi, K. Okamura, A. Takai, and A. Kotaki, ]. Biochem., 63, 814 (1968). 46K. Yagi, A. Kotaki, M. Naoi, and K. Okamura,J. Biochem., 60, 236 (1966). 47A. E. Bender and H. A. Krebs, Biochem..]., 46, 210 (1950). 48p. Handler, F. Bernheim, and J. R. Klein,.]. Biol. Chem., 138,203 ( 1941). 49M. Dixon and K. Kleppe, Biochim. Biophys. Acta, 96, 368 (1965). 5°A. H. Neims and L. Hellerman,J. Biol. ('hem., 237, PC 976 (1962). '~ K. Yagi, T. Ozawa, and M. N aoi, Biochim. Biophys. Acta, 185, 31 (1969). 5ZD. Wellner and H, Scannone, Biochemist~),, 3, 1746 (1964). 5:~K.Yagi and M. Nishikimi, ]. B iochem., 64, 371 (1968). '~4K. Yagi, T. Ozawa, and K. Okada, Biochim. Biophys. Acta, 35, 102 (1959). ~sW. T. Brown and P. (;. Scholefield, Proc. Soc. Expt. Biol. Med., 82, 34 (1953). 568. Edlbacher and (7). Wiss, Heh~. ('him. Acta, 27, 1831 (1944). ,~r.]. R. Klein and H. Kamin,J. Biol. Chem., 138, 507 ( 1941 ). ,sa(;. R. Bartlett,.]. Am. Chem. Sot., 70, 1010 (1948). ~u.]. R. Parikh, .]. P. (;reenstein, M. Winitz, and S. M. Birnbaum, .]. Am. Chem. Sot., 80, 953 (1958).
620
[2201
AMINO ACID OXIDATION
oxidation by c o m p e t i n g with the substrate. O n the o t h e r h a n d , aniline binds with the a p o e n z y m e by c o m p e t i n g with FADP 4 a n d p h e n o l s inhibit this reaction by c o m p l e x i n g with F A D ) a Riboflavin 5 ' - m o n o s u l f a t e a n d a d e n o s i n e 5 ' - m o n o s u l f a t e c o m p e t e with the F M N a n d A M P parts o f FAD, respectively, in c o m b i n i n g with the a p o e n z y m e . 6° p - C h l o r o m e r c u r i b e n z o a t e inhibits the oxidase in c o m p e t i t i o n with both the substrate a n d FAD. 61 S o m e d r u g s (e.g., p-aminosalicylic a c i d p 2 chlorp r o m a z i n e , 63 chlortetracycline, 64 c h l o r a m p h e n i c o l , 65 penicillin, 6~ streptomycin 65) show a c o m p l i c a t e d inhibitory m e c h a n i s m , i.e., s i m u l t a n e o u s o c c u r r e n c e o f two or t h r e e o f the a b o v e - m e n t i o n e d mechanisms. Fluorescence. T h e fluorescence o f the h o l o e n z y m e , w h e n excited with light at 450 m/x a n d m o n i t o r e d at 530 m/x, is less t h a n that o f free FAD. 66 W h e n the h o l o e n z y m e f o r m s a c o m p l e x with benzoate, f u r t h e r q u e n c h ing o c c u r s Y Optical Rotatory Dispersion (ORD) and Circular Dichroic (CD) Spectra. No significant differences in the values o f [odo a n d in helical content m e a s u r e d f r o m the t r o u g h a m p l i t u d e at 233 m/x are f o u n d a m o n g the apoe n z y m e , the h o l o e n z y m e , a n d the b e n z o a t e c o m p l e x o f this oxidase (Table II). 6"~8 TABLE I1 OPTICAL ROTATORY PROPERTIES OF D-AMINO ACID OXIDASE"
Oxidase species
[a]~5°
Helical contentt' (%)
Benzoate complex Holoenzyme Apoenzyme
--43 ° --47 ° --47 °
13 13 12
"Measured in buffer B. bCalculated from the amplitude of the trough at 233 m/x.
T h e h o l o e n z y m e a n d the b e n z o a t e c o m p l e x show a n o m a l o u s dispersion p a t t e r n s in the visible region d u e to the c o m b i n a t i o n o f the a p o e n z y m e with the c o e n z y m e a n d with benzoate. In the difference O R D 6°F. Egami and K. Yagi,J. Biochem., 43, 153 (1956). 61K. Yagi and T. Ozawa, Biochim. Biophys.Acta, 42, 381 (1960). 62K.Yagi, J. Okuda, T. Ozawa, and K. Okada, Biochem. Z., 528, 492 (1957). 63K.Yagi, T. Ozawa, and T. Nagatsu, Biochim.Biophys.Acta, 43, 310 (1960). 64K. Yagi, J. Okuda, T. Ozawa, and K. Okada, Biochim. Biophys. Acta, 34, 372 (1959). 65K.Yagi and T. Ozawa, Biochim.Biophys.Acta, 39, 304 (1960). 66V. Massey, B. Curti, and H. Ganther,J. Biol. Chem., 241, 2347 (1966). 67K. Yagi, T. Ozawa, and M. Harada, Syrup.Enzyme Chem.Japan, 14, 87 (1960). 68K.Yagi, M. Naoi, and A. Kotaki,J. Biochem., 59, 91 (1966).
[220]
D-AMINO
ACID OXIDASE
AND
ITS COMPLEXES
621
between the benzoate complex and the holoenzyme, an anomalous dispersion pattern having an inflection point at about 380 m/x (positive Cotton effect) is found. 4a [n an ORD pattern of the visible region, the purple intermediate shows a clear trough at 430 m/x, an inflection point close to 400 rntx, and a peak at 380 m~, but the semiquinoid enzyme gives a rather flat dispersion pattern./a The ORD pattern of the fully reduced enzyme shows slight anomalies in the range of 300-600 m/x.:" In CD spectra, 69 the holoenzyme shows two weak positive bands at 380 m/x ([0] : 11.4 deg.cm2/dM) and 435 m/x ([0] : 8.5 deg.cm"/dM), and tim benzoate complex shows only a high positive eltipticity centered at 377 m/x. In the case of the purple complex, a large negative band centered at 400 mix is found. The semiquinoid enzyme shows a CD pattern having only a slightly positive Cotton effect around 350 mlx. The CD spectrum of the fully reduced enzyme shows a broad negative band centered at 420 m/x and a positive band at 340 mtx (see Fig. I B). Oxidoreductio, States. Since the oxidoreduction equivalents of n-amino acid oxidase are 2, the existence of 3 oxidoreduction states, the oxidized (curve I1 in Fig. IA), the semiquinoid (curve IV in Fig. 1A), and the reduced (curve V in Fig. 1A) states are possible. However, the paramagnetic semiquinoid form of the enzyme is not the intermediate of the enzymatic reaction. The only demonstrable intermediate between the oxidized and the fully reduced states in the anaerobic reduction of the enzyme with substrate is the purple complex, which is practically diamagnetic la and shows a charge transfer band around 550 m/x (curve III in Fig. 1A). This complex converts into the semiquinoid enzyme upon aging in the dark. The conversion is characterized by the appearance of an absorption peak at 492 mtx (curve IV in Fig. I A), ~a the appearance of an electron spin resonance (ESR) signal and a decrease in the trough amplitude at 430 m/x in the ORD spectrum. This phenomenon is atlributed to the charge separation of the electrons shared by the enzyme and substrate moieties in the complex; the complex is assigned to be an inner complex. Molec,lm Weig41ls. The physicocbemical constants of the three species of the oxidase, the apoenzyme, the holoenzyme, and the benzoate complex, are shown in Table III. 6 From these values, the molecular weights of tim benzoate complex and the holoenzyme are calculated to be 112,000-115,000 and that of" the apoenzyme to be about 55,000. The N- and C-terminal analyses show that the enzyme consists of one polypeptide chain per 50,000 g, beginning at a methionine residue ~%. K¢~taki,N. Sugiura, and K. Yagi, Biochim.Biophys.Acta, 151,689 (1968).
622
AMINO ACID OXIDATION
[220]
TABLE III PHYSICOCHEMICAI. CONSTANTS OF D-AMINO ACtD OXIDASE
Constant
Benzoate complex
Holoenzyme
Apoenzyme
0 (Svedberg) S20,w D20.w ( 10-7 cm2/sec) V (ml/g) It/] (dl/g) Molecular weight ~
6.3 5.3 0.742" 0.033 11.20 × 104
7.1 5.8 0.026 11.56 x 104
3.6 6.2 0.730 5.46 x l04
"Determined by pycnometry. bCalculated from amino acid analysis. CCalculated from sedimentation and diffusion data.
and terminating at a leucine residue. 7° In addition, the minimal molecular weight calculated from the amount of bound FAD is 49,400, indicating that the benzoate complex and the holoenzyme exist in a stable dimeric form at least in the concentration range over 5 mg/ml, although the latter further associates in much higher concentration (above 20 mg/ml). On the other hand, the apoenzyme shows a different value for the molecular weight as a function of protein concentration; it behaves mainly as a dimeric form at high concentrations, and as a monomeric form at low concentrations, zl The purple intermediate sediments as a symmetrical 5.1 S boundary in the ultracentrifuge at 5° (protein concentration, 11 mg/ml), 13 suggesting that it is in a dimeric form. Amino Acid Composition. 7° The characteristic of the amino acid composition of the protein moiety of this oxidase is the high content of aromatic amino acids. This explains the high extinction coefficient of /El% the apoenzyme at 280 m/z ~ 2s0- 15.1). A large part of the dicarboxylic acids exist in the enzyme as the corresponding amides. The molecular weight of the enzyme calculated from the amino acid composition is 49,200. _
_
~°A. Kotaki, M. Harada, and K. Yagi,J. Biochem., 61,598 (1967). ~1K. Yagi, T. Ozawa, and N. Ohishi,J. Biochem., 64, 567 (1968).
AMINO ACID OXIDATION
[2201
Serine, phenylalanine, and tryptophan have quite high nonenzymatic rates. In the case of the latter, lowering the pyridoxal-P concentration to 1.5 × 10-4 M or less decreases the nonenzymatic reaction greatly with respect to that with peroxidase. 4 The increment ofdecarboxylation due to added enzyme is greatest with methionine as substrate, followed in order by serine, methionine sulfoxide, phenylalanine, alanine and tryptophan. Results obtained by Hill and Mann s using a similar system but containing p-cresol show that glycine, homoserine, aspartic acid, glutamic acid, ornithine, lysine, and asparagine are also decarboxylated. Kinetic Properties. The Km for DL-methionine is 7.8 × 10-4 M. Activity of Peroxidases from Other Sources. Enzyme preparations that have similar properties to the horseradish peroxidase system have been obtained from cabbage leaves, turnip root, squash fruits, and mustard leaves. An extract obtained by digitonin treatment of cell particulates is treated with (NH4)2SO4 to yield the enzyme fraction having the decarboxylase activity. The mustard and cabbage extracts when tested for peroxidase are highly active. A partially purified lactoperoxidase has also catalyzed the decarboxylation of methionine. 4V. M. Riddle and M. Mazelis, Plant Physiol., 40, 481 (1965). ~J. M. Hill and P.J.G. Mann, Biochem.J., 99, 454 (1966).
[220]
D-Amino Acid Oxidase I and Its Complexes (Hog Kidney) By KUNIO YAGI R CH C O O H + O2 + H~O
I
NH2
) R C C O O H + NHa + H2Oz
II
O
The preparation method reported by Negelein and Br6mel la'2 is based on isolation of the apoenzyme of this oxidase. Even though the preparation obtained by this classical method afforded fundamental knowledge about this enzyme, the sample itself was impure and unstable. Nowadays, this method has been almost completely replaced by
1EC 1.4.3.3; D-Amino acid:oxygen oxidoreductase (deaminating). laE. Negelein and H. Br6mel, Biochem. Z., 300, 925 (1939). 2K. Burton, Vol. II, p. 199.
[220]
D-AMINO ACID OXIDASE AND ITS COMPLEXES
609
n e w e r m e t h o d s 3-6 c h a r a c t e r i z e d by the use o f a stabilizer. Since the e n z y m e was f o u n d to f o r m a stable c o m p l e x with benzoate, z-'° it is most easily isolated in the f o r m o f an e n z y m e - b e n z o a t e c o m p l e x . T h e enz y m e - b o u n d b e n z o a t e can be easily e x c h a n g e d for the substrate; this c o m p l e x is a suitable starting material for the p r e p a r a t i o n o f free h o l o e n z y m e a n d its a p o e n z y m e . Since this c o m p l e x can be r e g a r d e d as an e n z y m e - s u b s t r a t e c o m p l e x model, al it should be strictly distinguished f r o m free h o l o e n z y m e . T h e reaction s e q u e n c e o f the a n a e r o b i c catalysis o f this e n z y m e is e x p r e s s e d as E + SH2 ~- E --SH2 ~- purple intermediate ~- EH.TS ~ EH~ + S w h e r e SH2 r e p r e s e n t s the substrate, a n d S the p r o d u c t . Since the p u r p l e i n t e r m e d i a t e c o m p l e x , '2 w h e n it accumulates, is s p o n t a n e o u s l y conv e r t e d into the s e m i q u i n o i d e n z y m e a n d the substrate radical, 13 the p r e p a r a t i o n o f the s e m i q u i n o i d e n z y m e is also described h e r e in addition to those o f the p u r p l e i n t e r m e d i a t e a n d the fully r e d u c e d forms, a l t h o u g h the s e m i q u i n o i d e n z y m e is not involved in the enzymatic reaction sequence.
Assay Method Principle. T h e e n z y m e activity has b e e n assayed by m e a s u r i n g , m a n o m e t r i c a l l y 2 or polarographically, 6,14 the rate o f o x y g e n cons u m p t i o n d u e to the oxidation o f substrate by this e n z y m e . Recently, a new assay m e t h o d was r e p o r t e d '5 in which the a m o u n t o f a m m o n i a liberated t h r o u g h the oxidative d e a m i n a t i o n o f substrate by this enz y m e is m e a s u r e d directly by the i n d o p h e n o l m e t h o d . Also, a m e t h o d based on the inactivation o f catalase by 3-amino-l,2,4-triazole in the
3F. P. Veitch and R. McComb,J. Am. Chem. Soc., 78, 1363 (1956). 4H. Kubo, T. Yamano, M. Iwatsubo, H. Watari, T. Soyama, J. Shiraishi, S. Sawada, N. Kawashima, S. Mitani, and K. Ito, Bull. Soc. Chim. Biol., 40, 431 (1958). 5V. Massey, G. Palmer, and R. Bennett, Biochim. Biophys. Acta, 48, I ( 196 t ). 6K. Yagi, M. Naoi, M. Harada, K. Okamura, H. Hidaka, T. Ozawa, and A. Kntaki, J. Biochem., 61,580 (1967). 7K. Yagi, T. Ozawa, and M. Harada, Nature, 184, 1938 (1959). SK. Yagi, T. Ozawa, and M. Harada, Nature, 188, 745 (1960). 9K. Yagi and T. Ozawa, Biochim. Biophys. Acto, 56, 413 (1962). '°K. Yagi and T. Ozawa, Biochim. Biophys. Acta, 56, 420 (1962). 11K.Yagi, Advances in Enz';molog3,, 27, 1 (1965). '2In aerobic catalysis, this intermediate reacts with oxygen. '3K. Yagi, K. Okamura, M. Naoi, N. Sugiura, and A. Kotaki, Biochim. Biophys. Acta. 146, 77 (1967). '*W. Kielley, Vol. VI, p. 272. 'ST. N agatsu and K. Yagi,J. Biochem., 60, 219 (1966).
610
AMINO ACID OXIDATION
[220]
p r e s e n c e o f H202 g e n e r a t e d by the a m i n o acid oxidase was r e p o r t e d for the detection o f a small a m o u n t o f oxidase activity. TM Usually, however, the p o l a r o g r a p h i c m e t h o d is r e c o m m e n d e d because o f its simplicity and rapidity. Procedure. T o 10 ml o f a m i x t u r e o f 0.1 M D-alanine 17 and 1.0 X 10 -5 M FAD in 0.1 M sodium p y r o p h o s p h a t e buffer, p H 8.3, 0.03-0.05 ml o f the e n z y m e solution (containing about 2 0 - 3 0 / x g o f e n z y m e protein) is a d d e d with constant mechanical stirring. T h e o x y g e n c o n s u m p t i o n is m e a s u r e d at 20 ° with a p o l a r o g r a p h i c o x y g e n electrode (e.g., Beckman O x y g e n Sensor) c o n n e c t e d to a r e c o r d e r . T h e e x p e r i m e n t a l arrangem e n t described by Kielley 14 is r e c o m m e n d e d . Enzymatic Activity and Specific Activity. O n e unit o f enzymatic activity is d e f i n e d as the a m o u n t that catalyzes the reaction resulting in the cons u m p t i o n o f 1 m i c r o m o l e o f o x y g e n per m i n u t e u n d e r the assay conditions. T h e specific activity is calculated as the activity per milligram o f protein. Protein is d e t e r m i n e d by the biuret method. TM
Purification Procedure Using benzoate as a stabilizer d u r i n g the purification p r o c e d u r e , a p u r e crystalline e n z y m e - b e n z o a t e c o m p l e x can be obtained f r o m hog kidney. This p r o c e d u r e was first devised by Kubo et al., 4 d e v e l o p e d by Massey et al., 5 and f u r t h e r i m p r o v e d by Yagi et alp F r o m this complex, h o l o e n z y m e and a p o e n z y m e can be p r e p a r e d . Purification can be carried out at r o o m t e m p e r a t u r e unless otherwise stated.
Purification and Crystallization of the Benzoate Complex of D-Amino Acid Oxidase Step 1. T h e capsule and fat are r e m o v e d f r o m hog kidney and discarded. After slicing, the red medulla is r e m o v e d and discarded; 250-g quantities o f sliced cortex are h o m o g e n i z e d in 1 liter o f buffer A TM in a W a r i n g B l e n d o r at half speed for the initial 30 seconds and at full speed for 2.5 minutes. F r o m 1 kg o f the cortex, 4.65 liters o f the h o m o g enate are obtained. T h e h o m o g e n a t e is adjusted to p H 7.6 by a d d i n g 1 N N a O H ; it is tLen slowly heated to 40 ° in a water bath set at 45 ° and 1ell. Scannone, D. Wellner, and A. Novogrodsky, Biochemistry,3, 1742 (1964). 17DL-Alanine can be used, because L-alanine does not affect the activity of this enzyme at this concentration. lSA. G. Gornall, C.J. Bardawill, and M. M. David, J. Biol. Chem., 177, 751 (1949); see also Vol. III, p. 450. ~aBufferA: 1.67 × 10-2 M sodium pyrophosphate buffer, pH 8.3, containing 0. 1% sodium benzoate.
[220]
D-AMINO ACID OXIDASE AND ITS COMPLEXES
611
maintained at 40 ° for 10 minutes. T h e n the h o m o g e n a t e is b r o u g h t to pH 5.2 by a d d i n g 1 N acetic acid, heated again to 40 °, and mainrained at 40 ° for 5 minutes. By centrifugation at 2200 g [br 10 minutes, a brownish red supernatant (ca. 3.6 liters) is separated from the pellet. Step 2. A m m o n i u m sulfate (176 g per liter) is a d d e d to the supernatant, and the mixture is allowed to stand for 1 h o u r at 5°. By centrifugation at 3000 g for 20 minutes, the precipitate is separated and dissolved in 100 ml of buffer A. T h e pH of this yellowish red solution is adjusted to 5.2 by the addition of 1 N acetic acid. T h e solution is heated to 55 ° in a water bath set at 60 °, maintained at 55 ° for 10 minutes, and is immediately centrifuged at 15,/)00 g for 10 minutes. A reddish yellow supernatant (ca. 115 ml) is obtained (first heat treatment). A m m o n i u m sulfate (114 g per liter) is a d d e d to the supernatant; after standing t o t 1 h o u r at 5°, the mixture is centrifuged at 15,000 g for 10 minutes and the s u p e r n a t a n t is discarded. T h e precipitate is dissolved in 50 ml of" buffer A. T h e solution is adjusted to pH 5.2 by adding 1 N acetic acid and then heated to 58 ° in a water bath set at 63 ° and mainrained at 58 ° for 3 minutes. After centrifugation o f the sample at 15,000 g for 10 minutes, the precipitate o f d e n a t u r e d protein is discarded. An orange yellow supernatant (ca. 50 ml) is obtained. By this second heat treatment, "° the concomitant catalase activity is usually eliminated. Step 3. A m m o n i u m sulfate (114 g per liter) is a d d e d to the supernatant, and after standing for 1 h o u r at 5°, the mixture is centrifuged at 15,000 g-for 10 minutes. T h e precipitate is dissolved in a minimal volume (ca. 10 ml) of buffer A. T h e enzyme solution is applied to a column of calcium phosphate gel-cellulose 't (2 x 15 cm) equilibrated previously with 0.05 M sodium phosphate buffer (pH 5.1). T h e enzyme is eluted with the same buffer; the enzyme moves down as a yellow band and is separated from brownish red impurities that remain at the upper part of the c o l u m n ) T h e eluate is r e c h r o m a t o g r a p h e d by the same procedure. T h e yield is 135 m g (see Table I). Step 4. T h e r e c h r o m a t o g r a p h e d eluate is b r o u g h t to 20% saturation with a m m o n i u m sulfate; after standing for 1 h o u r at 5°, the precipitated enzyme is separated by centrifuging at 15,000 g tot" 10 minutes and is dissolved in a minimal volume of buffer A. This solution is chilled in an
z°When a rapid preparation is n e e d e d , one can substitute h)r this step, a single heating (60 ° for 3 minutes), although the purity o f the p r e p a r a t i o n at this stage is somewhat lowered. alCalcium phosphate gel-cellulose: a mixture of calcium p h o s p h a t e gel =2 and cellulose p o w d e r ( 1: 15-30 w/w). 2'-'See Vol. 1, p. 98.
612
[220]
AMINO ACID OXIDATION
ice b a t h a n d a f e w d r o p s o f s a t u r a t e d a m m o n i u m
sulfate solution are
a d d e d to m a k e t h e s o l u t i o n slightly t u r b i d . A f t e r s t a n d i n g f o r s e v e r a l d a y s at 5 ° , t h e y e l l o w n e e d l e crystals w h i c h a p p e a r e d a r e c e n t r i f u g e d at 10,000 g f o r 10 m i n u t e s a n d d i s s o l v e d in b u f f e r A (ca. 7 m g / m l ) . T h e s o l u t i o n o f t h e c r y s t a l l i n e e n z y m e t h u s o b t a i n e d h as a b s o r p t i o n p e a k s at 3 7 9 m t t a n d 4 6 3 m/z a n d a m a r k e d s h o u l d e r at 4 8 6 m t t as s h o w n by c u r v e / i n Fig. 1A.
2.0
-~A
J~3
4I 300
B
400
III
500
3zI
I
0
400
500 Wovelength (mp.)
600
FiG. 1. Absorption and circular dichroic spectra of D-amino acid oxidase and its complexes. (A) Absorption spectra. (B) Circular dichroic spectra. Curves: 1, benzoate complex (the enzyme was mixed with 1 x 10-a M benzoate); 11, holoenzyme; 1H, purple intermediate (the enzyme was mixed with 5 x 10-2 M D-alanine in the presence of 1 × 10-j M lithium pyruvate and 5 × 10-z M ammonium sulfate); IV, semiquinoid enzyme complex with products (the enzyme was half-reduced with dithionite and was mixed with 3 x 10-2 M lithium pyruvate and 1 x 10-2 ~1 ammonium sulfate)'. V, fully reduced enzyme (the enzyme was reduced with 5 x 10-2 M D-alanine); VI, enzyme complex with Al-piperidine 2-carboxylate (the enzyme was mixed with 1 x 10-3 M this compound). The enzyme concentration was fixed at 1.2 × 10-4 M with respect to FAD. Measurements were made at room temperature. Unit of [0]: deg.cm2/dM.
Typical data for the overall purification procedure of the benzoate c o m p l e x a r e s u m m a r i z e d in T a b l e I.
[220]
D-AMINO
613
ACID OXIDASE AND ITS COMPLEXES
TABLE I SUMMARY OF PURIFICATION PROCEDURE OF BENZOATE COMPLEX OF D-AMINO ACID OXIDASE
Total volume (ml) Summary of procedure Step 1: Homogenate 4640 Crude extract 3640 Step 2: lst heat treatment 115 2nd heat treatment 52 Step 3: 1st column eluate 24 2nd column eluate 6.8 Step 5: Sephadex eluate .
Total activity (units)" Total KCN protein -- 10 n ~ l (mg) 860 740 710 11211 1150 930 . .
1720 1470 1420 1120 1150 930 .
182,000 26,000 1,370 760 200 135
Specific activity Recovery Degree (units/mg) of of KCN enzyme purifi-10 mM (%) cation 0.0047 0.028 0.52 1.47 5.8 6.9 8.8
0.0095 0.057 1.04 1.47 5.8 6.9 8.8
100 86 83 65 67 54 -
l 6 110 156 620 730 930
"A unit of activity is defined as the number of micromoles of oxygen consumed per minute.
The preparation obtained in step 4 is still too crude for physicochemical characterization; the following procedures are included for its further purification/a'24 Step 5. About 500 mg of the crystals of the benzoate complex obtained (usually from 10 kg of hog kidney slices) in step 4 is dissolved in 30 ml of buffer A and the solution is dialyzed against 3 liters of the same buffer for 24 hours at 5 °. The dialyzed solution is applied to a column of" Sephadex G-75 (2.5 x 35 cm), equilibrated previously with buffer A. The loading ratio is 10-20 mg of protein per gram of Sephadex. The enzyme is eluted with the same buffer, and yellow fractions having a ratio of about 1 mole of FAD per 50,000 g of protein are collected. Step 6. Crystallization of the benzoate complex fl'om the eluate obtained in step 5 is made in the same manner as described in step 4. Fine needle crystals are usually obtained, and after the preparation '-':~Further purification is also possible by repeating the crystallization 2-'~; times, though the yield becomes faMy low. '-'4As a simpliiied preparation of purified benzoate complex solution, the following procedure is recommended. 2~ The eluate from calcium phosphate gel-cellulose column is heated a~ 60 ° for 3 minutes at pH 5.2 and centrifuged at 15,000 g for 10 minutes. To the supernatant ammonium sulfate (I 1.4 g/100 ml) is added, and the mixture is centrifuged at 15,000 g for 10 minutes. The precipitate is dissolved in buffet" A (ca. 20 mg/ml). The solution containing about 500 mg of benzoate complex is applied to a column o! Sephadex (;-75 (5 × 40 crn) previously equilibrated with 0.01% aqueous solution of sodium benzoate and eluted with the same solution. Fractions having a ratio below 50,000 g of protein pet" mole of FAD are collected. The recovery is above 60% with respect to FAD. 2aK. Yagi, N. Sugiura, H. ()hama, and A. Kotaki,J. Biochem., 68,572 (1970).
614
AMINO ACID OXIDATION
[220]
has stood for several days, large hexagonal crystals appear in the solution. The protein moiety of this crystalline benzoate complex obtained from the Sephadex gel eluates is chemically homogeneous as j u d g e d by the N-terminal analysis. The crystals are a 1:1 complex composed of the oxidized enzyme (on the molecular basis of 50,000) and benzoateY 6
Preparation and Crystallization of the Holoenzyme 6 The holoenzyme is prepared from the crystalline benzoate complex by repeated expulsion of the benzoate from the complex with an excess of D-alanine. It is preferable to carry out this procedure in an ice bath. About 500 mg of the crystalline benzoate complex is dissolved in 50 ml of buffer B 27 (enzyme concentration:2 × 10 -4 M with respect to FAD) and mixed with 2 g of DL-alanine (final concentration 2.2 × 10 -1 M D-alanine); 6 g of solid ammonium sulfate is added to this solution (ca. 20% saturation). The pH of the mixture is adjusted to 6.1 by adding 1 N acetic acid. The purple precipitate formed is collected immediately by centrifuging at 15,000 g for 10 minutes. This treatment is repeated 3 times. The purple precipitate is dissolved in a small volume of buffer B (ca. 15 ml), and the solution is dialyzed overnight against 2 liters of the same buffer at 5 ° with mechanical stirring. The dialyzing medium is changed 2 times. A transparent deep yellow solution of the oxidized holoenzyme is obtained. To j u d g e the complete removal of benzoate, a splitting in the absorption peak at around 375 m/z is a good indication (see curve H in Fig. 1A, and also Absorption Spectra under Properties). To crystallize the holoenzyme, ammonium sulfate is added to the above-mentioned dialyzed preparation (enzyme concentration: ca. 6 × 10 -4 M with respect to FAD) to 1% saturation at 25 °. Then the pH is adjusted to 8.3. The solution is stored at 5 ° for about 10 days, and yellow hexagonal crystals are obtained. 28
Preparation of the Apoenzyme of D-Amino Acid Oxidase For the cleavage of the holoenzyme into the apoenzyme and FAD, the classical method according to Negelein and Br6meP a'2 has been widely used, a method which is also applicable to both the holoenzyme and its benzoate complex. However, the yield of the active apoenzyme is very low because of considerable denaturation due to t h e acid treatment. ~K. Yagi and M. Harada,J. Biochem., 57,463 (1965). 27Buffer B: 1.67 x 10-2M sodium pyrophosphate buffer, pH 8.3. ~SThe crystal of the holoenzyme can also be obtained by dialyzing the purple intermediate crystal against buffer B at 5°. 29 ~gK. Yagi and T. Ozawa,J. Biochem., 54, 202 (1963).
[220]
D-AMINO ACID OXIDASE AND ITS COMPLEXES
615
Recently, improved methods for the preparation of the apoenzyme, using column chromatography with hydroxylapatite 3° or with TEAEcellulose 6 as adsorbant, have been reported. A simpler method depends upon dialysis in the presence of a high concentration of KBr. a' Preparation of Apoenzyme by Dialysis. The benzoate-free holoenzyme (1 x 10 -4 M with respect to FAD) is dialyzed at 4 ° against a solution of 0.1 M sodium pyrophosphate, pH 8.5, containing 3 × 10-3 M EDTA and 1 M KBr. The volume inside the dialysis sac is 5 ml; the volume of the dialysis medium is 1 liter. The dialyzing medium is changed 3 times over a 2-day period until the yellow color of the holoenzyme disappears. The dialyzing medium is then changed to 0.1 M sodium pyrophosphate buffer, pH 8.5, and this dialysis is continued for 2 days to accomplish complete removal of KBr. The yield of the apoenzyme with respect to its enzymatic activity is nearly 100%. Preparation of Apoenz~me b~, Chromatography on TEAE-cellulose. Two milliliters of the holoenzyme solution in buffer B (protein concentration: 2 × 10 -4 M with respect to FAD) are applied to a TEAE-cellulose column (2.5 x 40 cm) equilibrated previously with 5 mM sodium phosphate buffer, pH 5.2. The loading ratio is about 1 mg of protein per gram of ion exchanger. The apoenzyme is eluted with the same buffer at 5° at a flow rate of 5 ml/30 minutes, and it appears in the effluent usually between fraction 10 and fraction 13 (each fraction volume, 5 ml). A yellow band of FAD is left at the upper part of the column.
Preparation of the Intermediate Complex and Its Related States
P'mple Intermediate Complex'a'3z,aa The purple complex, a reaction intermediate of D-amino acid oxidase, is crystallized from the mixture of the holoenzyme, the substrate, Dalanine, and the products, pyruvate and ammonia, under anaerobic conditions. The crystals consist of equimolar amounts of the enzyme (on the molecular basis of 50,000) and the substrate moiety which is readily converted into the products on aeration. Since concentration of the purple intermediate in the solution is governed by the concentrations of the substrate and the products, a4 a quantitative study should adopt definite concentrations of the enzyme, the substrate and the products, which are mixed under strictly anaerobic conditions. However, for the crystallization of the purple intermediate, the original method starting :"~Y. Miyake, K. Aki, S. Hashimoto, and T. Yamano, Bioehim. Biophys. Acta. 105, 86 (1965). a'V. Massey and B. Curti, ]. Biol. Chem., 241,3417 (1966). a2K. Yagi and T. Ozawa, Bioehim. Biophys. Acta, 60,200 (1962). :~:~K.Yagi and 'F. Ozawa, Biochim. Biophys. Acta, 81, 29 (1964). :~4K. Yagi, K. Okamura, N. Sugiura, and A. Kotaki, Biochim. Biophys. Acta, 159, 1 (1968).
616
AMINO ACID OXIDATION
[220]
from the enzyme-benzoate complex is recommended, because of its simplicity. In this case, some pyruvate is produced from D-alanine during the preparation procedure, and this product, together with the excess amount of another product, ammonia, added in the form of ammonium sulfate, affects the equilibrium to maintain the amount of the purple intermediate. The amount of pyruvate produced is sometimes sufficient, but in most cases it is necessary to add a small amount of powdered lithium pyruvate to make the solution deep purple; this is an adequate condition for crystallizing the purple intermediate. Crystallization Starting [rom the Enzyme-Benzoate Complex.a2,aa Pure crystalline enzyme-benzoate complex (0.2-1.0 g) is dissolved in 50 ml of buffer B, and 1 g of D-alanine is added with stirring. The pH of the solution is brought to 6.1 by adding 1 N acetic acid, and 6 g of ammonium sulfate is added to the solution. After standing at 5 ° for 30 minutes in an icebox, the precipitate is collected by centrifugation at 15,000 g for 10 minutes and dissolved in 50 ml of buffer B (final pH 7.5-7.7). Then D-alanine (about 1 g) is carefully added with stirring to the brownish yellow solution thus obtained, until the color of the solution turns to purple. The pH of the solution is brought to 6.1, and 6 g of ammonium sulfate is added. The purple colored precipitate is collected by centrifugation at 15,000 g for 10 minutes and is dissolved in a minimal volume of buffer B, so that the enzyme concentration becomes about 1 x 10-3 M with respect to FAD. The pH of the purple solution thus becomes 7.2, and the solution becomes about 5% saturated with respect to ammonium sulfate. If the pH is not 7.2, it should be adjusted exactly to this figure by the addition of acid or alkali. Powdered lithium pyruvate is carefully added to this solution until the solution becomes deep purple. The solution is transferred to a Thunberg-type tube filled with argon gas. At this moment, double refraction of flow is usually observed. After the preparation has stood at 5 ° overnight, a crop of purple crystals appears. The shape of the crystal is a hexagonal prism with bipyramids, a~ To check the conditions for crystallization, the following method is convenient; a small amount of the solution (0.02 ml) is placed on a piece of glass (such as a microscope slide) and sealed with petrolatum and a cover glass? ~ When the conditions are suitable, crystals form gradually and can be observed with a microscope. Crystallization Starting from the Holoenzyme. To obtain the purple intermediate solution, usually 3 ml of the holoenzyme (1.0 x 10 -4 M with respect to FAD dissolved in buffer B) is placed in the main chamber ~SK.Yagiand T. Ozawa,J. Biochem.,53, 162 ( !963).
[220]
D-AMINO
ACID OXIDASE
AND ITS COMPLEXES
617
o f a T h u n b e r g - t y p e cuvette, and D-alanine (5.0 X 10-=' M final concentration), lithium p y r u v a t e (1.0 x 10 -1 M final concentration), and a m m o n i u m sulfate (5.0 x 10 -2 M final concentration) are placed in the sidearm, v'~ T o eliminate oxygen, evacuation and flushing with p u r e argon ,gas are carried out, alternately, several times; the contents o f the sidearm are then a d d e d to the main c h a m b e r . A d e e p - p u r p l e solution (pH 7.0-7.2), having a s p e c t r u m shown by curve I11 in Fig. I A, is o b t a i n e d ? ~ If the c o n c e n t r a t i o n o f the e n z y m e is increased to ca. 5 x 10 -4 M and a m m o n i u m sulfate to 5 % saturation, crystals o f the p u r p l e i n t e r m e d i a t e can be obtained. Usually a longer time is r e q u i r e d fi)r the crystallization than for that in the case starting f r o m the enzymebenzoate complex. Semiqui,oid Form of D-Amino Acid Oxidase
A solution o f the semiquinoid e n z y m e can be f o r m e d r a t h e r easily by anaerobic t r e a t m e n t with sodium dithionite 37 or by strong light irradiation 3s'39 of the h o l o e n z y m e u n d e r anaerobic conditions. On the o t h e r hand, the p u r p l e intermediate is c o n v e r t e d slowly into the semiquinoid e n z y m e u p o n storage in the dark. ~3 This conversion is accelerated by illumination. F r o m this solution, the semiquinoid f o r m o f the e n z y m e can be crystallized. 4°'4~ T h r e e milliliters o f the p u r p l e complex solution (ca. 2 x 10-4 M with respect to FAD) in a T h u n b e r g - t u b e , are illuminated by a 30 W tungsten lamp (e.g., Mazda daylight lamp) at a distance o f 20 cm for 48 hours at 5 ° u n d e r anaerobic conditions. T h e solution gradually turns to reddish purple. After the absorption s p e c t r u m reaches curve IV in Fig. I A, p o w d e r e d a m m o n i u m sulfate is a d d e d anaerobically to the solution until the c o n c e n t r a t i o n is 0.2 M (ca. 5 % saturation) and its p H is adjusted to 6.8-7.0. W h e n this solution is stored at 5 ° for 2 days, reddish b r o w n crystals appear. T h e crystal is a h e x a g o n a l prism with bipyramids. In the crystalline p r e p a r a t i o n , this semiquinoid e n z y m e exists as its c o m p l e x with coexisting substrate, D-alanine, a n d / o r products, pyruvate and a m m o n i a , t h o u g h these c o m p l e x e s are not involved in the enzymatic reaction sequence. :~qn this case, the purple intermediate present reaches 90% of its maximal fi)rmation? 4 :~T'I'.Nakamura, S. Nakamura, and Y. Ogura,J. Biochem., 54, 512 ( 1963). aSH. Watari, K-J. Hwang, K. Ashida, and K. Kinoshita, Biochim. Biophys. Acta, 128, 256 (1966). agV. Masseyand G. Palmer, Biochemistry, 5, 3181 (1966). 4°K. Yagi and N. Sugiura,J. Biochem., 60, 738 (1966), ~ K. Yagi, N. Sugiura, K. Okamura, and A. Kotaki, Biochim. BiophU. Acta, 151,343 (1968).
618
AMINO ACID OXIDATION
[220]
Crystallization of the Fully Reduced Form of D-Amino Acid Oxidase 34"42 The fully reduced form of this oxidase is produced by anaerobic reduction of the enzyme with excess D-alanine. Its crystallization can be performed by increasing the concentration of ammonium sulfate. The crystallization procedure is somewhat similar to that of the purple intermediate starting from the holoenzyme except for the absence of pyruvate. The key point of the preparation is, therefore, to carry out the procedure under strictly anaerobic conditions, which enables one to obtain the fully reduced form without significant contamination of pyruvate. To 6 ml of the enzyme solution containing 200 mg of the holoenzyme (6.7 x 10 -4 m with respect to FAD), 160 mg of powdered D-alanine (3 X 10-1 M final concentration) is added with gentle shaking under anaerobic conditions. The yellow color of the holoenzyme solution rapidly changes into purple and then gradually turns to a pale yellow, indicating that the enzyme is fully reduced. To this pale yellow solution, 168 mg of powdered ammonium sulfate (2.3 x 10 -1 M final concentration, ca. 5% saturation) is added with stirring and the resulting solution is stored at 5 ° under anaerobic conditions (gas phase, argon). After storing this solution over a week, a crop of pale yellow crystals appears. The shape of the crystals is a hexagonal prism, or a hexagonal prism with bipyramids. The crystals are a 1:1 complex composed of the fully reduced enzyme (on a molecular basis of 50,000) and the intact substrate, D-alanine.
Properties Absorption Spectra. The visible absorption spectrum of the holoenzyme is shown by curve H in Fig. 1A. The marked splitting of the peak at around 375 mft is characteristic. 43 When the enzyme combines with benzoate, the splitting of the absorption peak disappears with some hypochromism and a three-banded structure appears at an absorption peak around 460 m~ 43 (see curve I in Fig. 1A). The molar extinction coefficients for the holoenzyme and the benzoate complex at their absorption peaks, 455 m/.t and 463 m/~, respectively, are approximately 1.13 X 104. The extinction coefficient (E2s0) 1~ of the apoenzyme is 15.12 The purple intermediate complex is characterized by its broad absorption band in the vicinity of 550 mft (curve III), 13 whereas the semiquinoid enzyme is characterized by its absorption peak at 492 m/~ ( c u r v e IV). 3"&39"4°'41 On the other hand, the fully reduced form of the enzyme has no absorption peak in the visible wavelength region (curve 42K. Yagi and K. Okamura, Biochem. Biophys. Res. Commun., 21,399 (1965). 43A. Kotaki, M. Naoi, and K. Yagi,J. Biochem., 59, 625 (1966).
[220]
D-AMINO ACID OXIDASE AND ITS COMPLEXES
619
V). 34'42 Curve VI shows the spectrum of the complex of the oxidized enzyme with Al-piperidine 2-carboxylate, 44 which is formed from spontaneous cyclization of a-oxo-E-aminocaproate produced from Dlysine by this enzyme. 45 This spectrum is also characterized by a broad absorption band at the longer wavelength region, which is considered to be a charge transfer band. 44"46 A similar charge transfer band can also be observed by mixing the enzyme with other n-donors, such as 0-aminobenzoate, pyrroline 2-carboxylate, and indole 2-carboxylate. 44 Substrate Spec!ficitv. Although the substrate commonly used for study is D-alanine, in which case the molecular activity 6 (with respect to the molar concentration of FAD, measured by routine polarographic procedure at 20 °) is 435, the enzyme oxidizes various D-a-amino acids 47 and N-monoalkylated D-a-amino acids? 8 However, acidic D-a-amino acids are not oxidized. 49 Enzymatic oxidation of D-lysine is interrupted immediately by Al-piperidine 2-carboxylate, a strong inhibitor, [brmed from the reaction product through spontaneous cyclization. 4"~Glycine, ~° o-a-hydroxy acids (e.g., D-lactic acid), 51 and L-proline ~2,'~a are reported to be attacked by this enzyme at a slower rate. Electro~ Acceptors. Methylene blue and 2,6-dichlorophenolindophenol can serve as hydrogen acceptors, though they are not as active as molecular oxygen. 49 Inhibitors. Systematic investigation of the mechanism of inhibition using benzene derivatives 54 revealed that 0-COOH, ¢-NH2, and O-OH functions are responsible for competition with the substrate, competition with FAD, and combination with FAD, respectively. In accord with this indication, various carboxylic acids, including fatty acids, '~'~ some L-amino acids, 4"~,56benzoic acid, 57 and its derivatives, 54,.58and heterocyclic carboxylic acids (e.g., pyrrole 2-carboxylic acidSa), inhibit enzymatic 44V. Masse), and H. (;anther, Biockemist~)., 4, 1161 (1965). 4'5K. Yagi, K. Okamura, A. Takai, and A. Kotaki, ]. Biochem., 63, 814 (1968). 46K. Yagi, A. Kotaki, M. Naoi, and K. Okamura,J. Biochem., 60, 236 (1966). 47A. E. Bender and H. A. Krebs, Biochem..]., 46, 210 (1950). 48p. Handler, F. Bernheim, and J. R. Klein,.]. Biol. Chem., 138,203 ( 1941). 49M. Dixon and K. Kleppe, Biochim. Biophys. Acta, 96, 368 (1965). 5°A. H. Neims and L. Hellerman,J. Biol. ('hem., 237, PC 976 (1962). '~ K. Yagi, T. Ozawa, and M. N aoi, Biochim. Biophys. Acta, 185, 31 (1969). 5ZD. Wellner and H, Scannone, Biochemist~),, 3, 1746 (1964). 5:~K.Yagi and M. Nishikimi, ]. B iochem., 64, 371 (1968). '~4K. Yagi, T. Ozawa, and K. Okada, Biochim. Biophys. Acta, 35, 102 (1959). ~sW. T. Brown and P. (;. Scholefield, Proc. Soc. Expt. Biol. Med., 82, 34 (1953). 568. Edlbacher and (7). Wiss, Heh~. ('him. Acta, 27, 1831 (1944). ,~r.]. R. Klein and H. Kamin,J. Biol. Chem., 138, 507 ( 1941 ). ,sa(;. R. Bartlett,.]. Am. Chem. Sot., 70, 1010 (1948). ~u.]. R. Parikh, .]. P. (;reenstein, M. Winitz, and S. M. Birnbaum, .]. Am. Chem. Sot., 80, 953 (1958).
620
[2201
AMINO ACID OXIDATION
oxidation by c o m p e t i n g with the substrate. O n the o t h e r h a n d , aniline binds with the a p o e n z y m e by c o m p e t i n g with FADP 4 a n d p h e n o l s inhibit this reaction by c o m p l e x i n g with F A D ) a Riboflavin 5 ' - m o n o s u l f a t e a n d a d e n o s i n e 5 ' - m o n o s u l f a t e c o m p e t e with the F M N a n d A M P parts o f FAD, respectively, in c o m b i n i n g with the a p o e n z y m e . 6° p - C h l o r o m e r c u r i b e n z o a t e inhibits the oxidase in c o m p e t i t i o n with both the substrate a n d FAD. 61 S o m e d r u g s (e.g., p-aminosalicylic a c i d p 2 chlorp r o m a z i n e , 63 chlortetracycline, 64 c h l o r a m p h e n i c o l , 65 penicillin, 6~ streptomycin 65) show a c o m p l i c a t e d inhibitory m e c h a n i s m , i.e., s i m u l t a n e o u s o c c u r r e n c e o f two or t h r e e o f the a b o v e - m e n t i o n e d mechanisms. Fluorescence. T h e fluorescence o f the h o l o e n z y m e , w h e n excited with light at 450 m/x a n d m o n i t o r e d at 530 m/x, is less t h a n that o f free FAD. 66 W h e n the h o l o e n z y m e f o r m s a c o m p l e x with benzoate, f u r t h e r q u e n c h ing o c c u r s Y Optical Rotatory Dispersion (ORD) and Circular Dichroic (CD) Spectra. No significant differences in the values o f [odo a n d in helical content m e a s u r e d f r o m the t r o u g h a m p l i t u d e at 233 m/x are f o u n d a m o n g the apoe n z y m e , the h o l o e n z y m e , a n d the b e n z o a t e c o m p l e x o f this oxidase (Table II). 6"~8 TABLE I1 OPTICAL ROTATORY PROPERTIES OF D-AMINO ACID OXIDASE"
Oxidase species
[a]~5°
Helical contentt' (%)
Benzoate complex Holoenzyme Apoenzyme
--43 ° --47 ° --47 °
13 13 12
"Measured in buffer B. bCalculated from the amplitude of the trough at 233 m/x.
T h e h o l o e n z y m e a n d the b e n z o a t e c o m p l e x show a n o m a l o u s dispersion p a t t e r n s in the visible region d u e to the c o m b i n a t i o n o f the a p o e n z y m e with the c o e n z y m e a n d with benzoate. In the difference O R D 6°F. Egami and K. Yagi,J. Biochem., 43, 153 (1956). 61K. Yagi and T. Ozawa, Biochim. Biophys.Acta, 42, 381 (1960). 62K.Yagi, J. Okuda, T. Ozawa, and K. Okada, Biochem. Z., 528, 492 (1957). 63K.Yagi, T. Ozawa, and T. Nagatsu, Biochim.Biophys.Acta, 43, 310 (1960). 64K. Yagi, J. Okuda, T. Ozawa, and K. Okada, Biochim. Biophys. Acta, 34, 372 (1959). 65K.Yagi and T. Ozawa, Biochim.Biophys.Acta, 39, 304 (1960). 66V. Massey, B. Curti, and H. Ganther,J. Biol. Chem., 241, 2347 (1966). 67K. Yagi, T. Ozawa, and M. Harada, Syrup.Enzyme Chem.Japan, 14, 87 (1960). 68K.Yagi, M. Naoi, and A. Kotaki,J. Biochem., 59, 91 (1966).
[220]
D-AMINO
ACID OXIDASE
AND
ITS COMPLEXES
621
between the benzoate complex and the holoenzyme, an anomalous dispersion pattern having an inflection point at about 380 m/x (positive Cotton effect) is found. 4a [n an ORD pattern of the visible region, the purple intermediate shows a clear trough at 430 m/x, an inflection point close to 400 rntx, and a peak at 380 m~, but the semiquinoid enzyme gives a rather flat dispersion pattern./a The ORD pattern of the fully reduced enzyme shows slight anomalies in the range of 300-600 m/x.:" In CD spectra, 69 the holoenzyme shows two weak positive bands at 380 m/x ([0] : 11.4 deg.cm2/dM) and 435 m/x ([0] : 8.5 deg.cm"/dM), and tim benzoate complex shows only a high positive eltipticity centered at 377 m/x. In the case of the purple complex, a large negative band centered at 400 mix is found. The semiquinoid enzyme shows a CD pattern having only a slightly positive Cotton effect around 350 mlx. The CD spectrum of the fully reduced enzyme shows a broad negative band centered at 420 m/x and a positive band at 340 mtx (see Fig. I B). Oxidoreductio, States. Since the oxidoreduction equivalents of n-amino acid oxidase are 2, the existence of 3 oxidoreduction states, the oxidized (curve I1 in Fig. IA), the semiquinoid (curve IV in Fig. 1A), and the reduced (curve V in Fig. 1A) states are possible. However, the paramagnetic semiquinoid form of the enzyme is not the intermediate of the enzymatic reaction. The only demonstrable intermediate between the oxidized and the fully reduced states in the anaerobic reduction of the enzyme with substrate is the purple complex, which is practically diamagnetic la and shows a charge transfer band around 550 m/x (curve III in Fig. 1A). This complex converts into the semiquinoid enzyme upon aging in the dark. The conversion is characterized by the appearance of an absorption peak at 492 mtx (curve IV in Fig. I A), ~a the appearance of an electron spin resonance (ESR) signal and a decrease in the trough amplitude at 430 m/x in the ORD spectrum. This phenomenon is atlributed to the charge separation of the electrons shared by the enzyme and substrate moieties in the complex; the complex is assigned to be an inner complex. Molec,lm Weig41ls. The physicocbemical constants of the three species of the oxidase, the apoenzyme, the holoenzyme, and the benzoate complex, are shown in Table III. 6 From these values, the molecular weights of tim benzoate complex and the holoenzyme are calculated to be 112,000-115,000 and that of" the apoenzyme to be about 55,000. The N- and C-terminal analyses show that the enzyme consists of one polypeptide chain per 50,000 g, beginning at a methionine residue ~%. K¢~taki,N. Sugiura, and K. Yagi, Biochim.Biophys.Acta, 151,689 (1968).
622
AMINO ACID OXIDATION
[220]
TABLE III PHYSICOCHEMICAI. CONSTANTS OF D-AMINO ACtD OXIDASE
Constant
Benzoate complex
Holoenzyme
Apoenzyme
0 (Svedberg) S20,w D20.w ( 10-7 cm2/sec) V (ml/g) It/] (dl/g) Molecular weight ~
6.3 5.3 0.742" 0.033 11.20 × 104
7.1 5.8 0.026 11.56 x 104
3.6 6.2 0.730 5.46 x l04
"Determined by pycnometry. bCalculated from amino acid analysis. CCalculated from sedimentation and diffusion data.
and terminating at a leucine residue. 7° In addition, the minimal molecular weight calculated from the amount of bound FAD is 49,400, indicating that the benzoate complex and the holoenzyme exist in a stable dimeric form at least in the concentration range over 5 mg/ml, although the latter further associates in much higher concentration (above 20 mg/ml). On the other hand, the apoenzyme shows a different value for the molecular weight as a function of protein concentration; it behaves mainly as a dimeric form at high concentrations, and as a monomeric form at low concentrations, zl The purple intermediate sediments as a symmetrical 5.1 S boundary in the ultracentrifuge at 5° (protein concentration, 11 mg/ml), 13 suggesting that it is in a dimeric form. Amino Acid Composition. 7° The characteristic of the amino acid composition of the protein moiety of this oxidase is the high content of aromatic amino acids. This explains the high extinction coefficient of /El% the apoenzyme at 280 m/z ~ 2s0- 15.1). A large part of the dicarboxylic acids exist in the enzyme as the corresponding amides. The molecular weight of the enzyme calculated from the amino acid composition is 49,200. _
_
~°A. Kotaki, M. Harada, and K. Yagi,J. Biochem., 61,598 (1967). ~1K. Yagi, T. Ozawa, and N. Ohishi,J. Biochem., 64, 567 (1968).