Crystallization and some properties of D -amino acid oxidase apoenzyme

86 BBA

BIOCHIMICA ET BIOPHYSICA ACTA

65224

CRYSTALLIZATION AND SOME PROPERTIES OF D-AMINO ACID OXIDASE APOENZYME

YOSHIHIRO MIYAKE, KEN]I AKI', SUMIKO HASHIMOTO' AND TOSHIO YAMANO Department of Biochemistry, School of Medicine, Osaka University, Osaka (Japan) (Received November roth, 1964)

SUMMARY

A method for the preparation of n-amino acid oxidase (n-amino acid: O 2 oxidoreductase, EC 1+3.3) apoenzyme in a crystalline form is described. The preparation was completely free from flavin-adenine dinucleotide and was stable for at least a month in the frozen state. The values of S020'W and D0 20 ,w of the apoenzyme obtained were 8.1' ro- 13 sec and 6.0' 10- 7 cm 2jsec respectively, and from these constants the molecular weight of the apoenzyme was calculated as 125 000, assuming that the partial specific volume was 0.74- The apoenzyme was found to bind two moles of flavin-adenine dinucleotide per mole. The presence of two different types of SH groups in the enzyme was demonstrated spedrophotometrically with pchloromercuribenzoate. As a result, approximately 2 moles of SH groups reacted rapidly with p-chloromercuribenzoate and the other 8 reacted slowly. Inhibition of the enzyme by p-chloromercuribenzoate was partially non-competitive.

INTRODUCTION

Since the time when D-amino acid oxidase (n-amino acid: O2 oxidoreductase, EC 104.3.3) apoenzyme was first purified by NEGELEIN AND BROMEL 1 , their method has been widely used for the preparation of the apoenzyme of flavin enzymes. Recently, KUBO et al.2 succeeded in the crystallization of n-amino acid oxidase, while MASSEY ei al. 3 ,4 have also reported a method for the crystallization of the iron-free enzyme with very high specific activity. YAGI AND OZAWA 5 have shown that these preparations contained benzoic acid which was used to stabilize the enzyme during the purification. The apoenzyme prepared by NEGELEIN AND BROMEL showed a lower specific activity and was obtained in lower yield than the holoenzyme purified by MASSEY et al.": Moreover, their apoenzyme was relatively unstable even on storage at _20 0 (ref. 6). The present paper reports a method for purification and crystallization of the apoenzyme completely free from FAD, using hydroxylapatite Abbreviations: FeMB, p-chloromercuribenzoate. • Department of Physiology, School of Medicine, Tokushima University, Tolcuahima (Japan).

Biocliim, Biophys, Acta, 105 (1965) 86-99

CRYSTALLlZATION OF D-AMINO ACID OXIDASE APOENZYMD for separation of the holoenzyme from the apoenzyme and dialysis for releasing FAD6. With the result ant crystalline apoenzyme, the aut hors have attempt ed t o confirm reported data, such as the values of the sediment ation and diffusion constants of the apoenzyme and sp ectral characteristics of the reconstituted holoenzyme reported by YAGI et al .~, 7 . Detailed investigatio ns a ll the numbers of FAD which can bind to the ap oenzyme, and on the nature of SH gronps in the enzyme are also described. Concerning the binding of FAD, MASSEY et al. 4 have already reported th at I mole of FAD corresponded t o a minimum molecular weight 45 700. YAGI AND OZAWA 8 ha ve also r eported th at 2 moles of FAD are bound to I mole of the enzyme. Both results were obtained with the holoenzyme. On the nature of the SH groups in the enzyme, K u ao et al.2 have reported that 6 SH groups per mole of enzyme were detectable by amperomet ric analysis, and I of these !,TfOUpS is concerned with the binding of FAD . YAGI AND OZAWAo have also shown kinetically that SH groups in the enzyme are concern ed with the binding of the adenylic moiety of FAD. Thes e two properties, namely the numbers of bound FAD and the nature of the SH groups in the enzyme, are very impo rtant for elucidating the mechanism of the enzyme action , especially in analysis of th e present evidence on th e mechanism of enzyme acti on, which was shown by KUBo et al.1O, NAKAMURAet al.", YAMAN0 12 and MASSEy13 • MATERIALS AND METHODS Pig kidneys were used as a source of the enzyme . Catalase (EC 1.11.1.6) was crys tallized from beef liver by the pr ocedure of SHIRAKAWA14 • Crystalline trypsin (E C 3.4+4) and a bacterial proteinase from Bacillus subt-ilis, Nagarse, were purchased from Mochida Pharmaceuti cal Company, Tokyo Japan, and Nagase Indu strial Company, Osaka J apan, respectively. FAD was obtained from Wako Ph armaceutical Company , Tokyo Japan. All other chemicals were of anal ytic al grade. During the purification, th e enzymic activity was assayed manometrically at 38° under th e st andard conditions described by BURTON 15• In other experiments, the enzymic activity was assayed by the polarograph ic method without catalase according to the meth od of CHANCE AND WILLIAMS16 • For kinetic studies on the inhibition by P CMB, the manom etric method was used. However, the conditions used differed from those of BURTON. Thus, reactions were all carried out at 25° and cat alase was not used. P CME was placed in one of the side arms and poured into the main chambers 3 min before the adclition of nt-alanine to the enzyme solution. Enzyme-bound FAD was assayed both by titration of FAD with substrate and by dialysis equilibrium between FAD and apoenzyme. For the titration, vari ous concentrations of FAD were added to the apoenzyme solutions, and the absorbancies at 450 mil- were measured 30 min after th e addition of F AD. Then 0 .2 ml of 1M DLalanin e was rapidly mix ed with th e enzyme solutions using an adder-mixer of BOYER'S t ype! ", and immediately liquid paraffin wasint roduced over th e reaction mixture. After st anding the soluti on for 30 min , its absorbancy at 450 mp. was measured. Dialysis equili brium was carried out as follows ; a sample of the apoenzyme solution was dialysed exte nsively wit h st irring for 24 h against 0.1 M pyrophosph at e buffer (pH 8.3), plu s 50 p.M FAD, the dialysis vessel being kept completely in th e dark. Th e concentrati ons of FAD inside and out side of th e dialysis sac were then deterBiochim. Biophys. A cta, 105 (1965) 86- 99

88

Y. MIYAKE

et al.

mined spectrophotometrically at 450 mth for FAD and at 455 mth for the enzymebound FAD using the BruM-value for FAD of II.31·mole-l·cm-l at 450 mth (ref. 18). The protein concentration was determined by the biuret method-", by the microKjeldahl method and by direct weighing. SH groups in the enzyme were determined spectrophotometrically by the procedure of BOYER 29, i.e. the apoenzyme was mixed with PCMB in 0.1 M pyrophosphate buffer (pH 8.3), and the change in the absorbancy at 250 mth was followed. The concentration of PCMB was determined spectrophotometrically, using an BruMvalue of I6.gl·mole- 1 . cm- 1 at 232 mft (ref. 20). For reconstitution of the holoenzyme, the apoenzyme was dissolved in 0.1 M pyrophosphate buffer (pH 8.3), and excess FAD was added to the solution. After standing the solution for 30 min at room temperature, it was carefully adjusted to pH 6.3 with 2 N acetic acid. Next, (NH
CRYSTALLIZATION OF D-AMINO ACID OXIDASE APOENZYME

8g

against protein concentrations. The plots were then extrapolated to zero concentration of the protein. RESULTS

Preparation of the apoenzyme I kg of pig kidney was sliced, and fat, connective tissue, and medulla were discarded. Sliced kidney cortex was homogenized in 31 of 16 mM pyrophosphate buffer (pH 8.3). Step I: The homogenate was incubated at 30° for 40 min. Step 2: The pH of the homogenate was adjusted to 5.6 by the careful addition of 2 N acetic acid. The temperature of the homogenate was then increased to 38° and maintained at this temperature for 5 min. The homogenate was then rapidly cooled to below 10° by immersion in ice-water, and the homogenate was centrifuged at IS 000 X g for IS min. The supernatant was carefully separated from the loose pellet and the precipitate was discarded. Solid (NH,1)2S0'1 (175 gil) was added to the supernatant. Then the solution was recentrifuged at IS 000 X g for IS min. The precipitate was dissolved in 500 ml of 25 mM sodium phosphate buffer (pH 7.0). Subsequent steps were all carried out at about 5°. Step 3: The enzyme solution was centrifuged at 50 000 X g for go min in a Hitachi Model 40P refrigerated preparative centrifuge. After centrifugation, the precipitate was discarded and (NH4)2S04 (14 g per IOO ml) was added to the supernatant. The solution was centrifuged at 7000 X g for 10 min, and the precipitate was dissolved in ISO ml of 25 mM sodium phosphate buffer (pH 7.0). Step 4: The enzyme solution was placed in dialysis sac and dialysed for 24 h with stirring against the same buffer containing 175 g of (NH4)2S04 per 1. The outside solution was changed several times. After dialysis, the sample was centrifuged and the supernatant was discarded. The precipitate was then dissolved in 20 mM sodium phosphate buffer (pH 6.3). Step 5: The enzyme solution was dialysed with stirring against 20 mM sodium phosphate buffer (pH 6.3), for 12 h. A slight precipitate appeared, which was discarded by centrifugation, and the supernatant was poured into a solution of 20 ruM sodium phosphate buffer (pH 6.3), containing hydroxylapatide. The mixture was stirred for 30 min, and then centrifuged at 1500 X g for 10 min. The precipitate of hydroxylapatite was resuspended in 0.1 M sodium phosphate buffer (pH 6.3), and stirred for 30 min. The hydroxylapatite was removed by centrifugation at 7000 X g for 10 min and (NH4)2S04 (20 g per 100 ml) was added to the clear supernatant. The enzyme solution was centrifuged at 7000 X g for 10 min and the supernatant was discarded. The precipitate was dissolved in 20 mM sodium phosphate buffer (pH 6.3). Step 6: The enzyme solution was dialysed against the same buffer with stirring, overnight. A slight precipitate appeared. which was centrifuged off and the solution was applied to a hydroxylapatite cellulose column buffered with 20 ruM sodium phosphate buffer (pH 6.3). The column was then washed with the same buffer until the absorbancy of the eluate at 280 mf/" which appeared during this washing, had completely disappeared. The apoenzyme fractions were then eluted with 60 mM sodium phosphate buffer (pH 6.3), leaving a pale brownish-yellow band at the top of the column (Fig. I). The eluates containing the enzymic activity were collected Biochim, Biophys. Acta, 105 (1965) 86-99

go

Y. MIYAKE

et al,

r - - - O.02 M

=<.

E

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1.0

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80

20

Fig. I . H y drox y la patrte-cellu lose column chromatogr ap hy of the apoenzy m e with sodium p hosphate buffer (pH 6.3). 200 m l of hydroxylap at it e b u ffered with 20 mM sod iu m phospha t e buffer (pH 6.3 ), wa s mixed with 10 g of ce llulose pow der immediately before use. The column (limensions were 3 em X 20 em and the volume of the fractions was 10 ml, 0-0, enzymic activity after the addition of FAD; x - X, absorbancy at 280 mIl of each solution.

and the apoenzyme, precipitated by the addition of (NH4)2S0" (20 g per 100 ml), was obtained by centrifuging t he mixture at 7000 X g for 10 min . The apoenzyme was dissolved in a minimum volume of 20 mM sodium phosphate buffer (pH 6.3), approximately in the proportion of 40 mg/ml. Step 7: T he apoenzyme solution was dialysed against the same buffer with st irring for 2 h and then left at 0° to allow crystallization. Table I shows the details of a t ypi cal prepar at ion . TABLE I SUMMARY OF THE PURIFICATION OF THE APOENZYME

All figures in the column were of the solutions that were fractionated by (NH.).SO. after each treatment. I u nit is defined a s III of 0. uptake per m in .

Steps

(I) (2) (3) (4)

Homogenate Acid and h ea t treatment Centrifugation (50 000 X g. 90 min) Dialysis against (NH.l.SO.containing buffer solution (5) Hydroxylapatite adsorption (batchwise) (6) Hydroxylapatite-cellulose column chromatography (7) Crystals collected by centrifugation and disso lved in 20 mM sodium phosphate buffer (pH 6.3)

Biochim , B iop hy s. A cia, 105 (1965) 86-99

Specific acti vity (unitslmg}

Total volume (ml)

Total pl'otein ( mg)

T otal units

Yield

4000 610 116

138 800 1018 7 2447

119 880 53540 45 °7 8

0·79 5·27 18.3 8

44 ·7 37. 6

98

1 ° 42

355 89

31.16

29·7

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262

16 557

63. 08

13.8

3

46

839 2

182.00

7. 1

2

33

6600

200.00

5·5

(%)

CRYSTALLIZATION OF D-AMINO ACID OXIDASE APOENZYME

Fig.

2.

Photomicrograph of crystals of the apoenzyme. Magnification X

91

1000.

Properties of the crystalline apoenzyme The apoenzyme was crystallized as very fine needles (Fig. 2), which when disso1ved gave a clear and colourless solution. No FAD was detectable by fluorimetric analysis following the method of BESSEY et al. 22 .or by spectrophotometric analysis. In the absence of FAD, IO,uM apoenzyme did not have any enzymic activity. The slight contamination of the apoenzyme with holoenzyme, which still present after extensive dialysis, was completely removed by hydroxylapatite treat10-

'c"

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500

Wavelength (m,u)

Fig. 3. Absorption spectrum of the apoenzyme and the reconstituted holoenzyme in 0.1 M pyrophosphate buffer (pH H.]). I, apoenzyme; 2, holoenzyme; 3, holoenzyme reduced with DLalanine. 0.65 mg of the apoenzyme per ml was used for measurement of the ultraviolet spectrum, and 3.] mg per ml for that of the visible spectrum. Solutions were reduced by addition of o. I ml of I M m.-alaninc under anaerobic conditions. The total volume of the solutions was 3.0 m1.

Biochim. Biopbys. Acta, 105 (1965) 86-99

Y. MIYAKE

et al

ment. The holoenzyme was elut ed with 20 mM sodium phosphate buffer (pH 6.3), while the apoenzyme remained adsorbed to the hydroxylapatite. The crystalline apoenzyme had a specific activity of 200,u1 02fmin per mg protein in the presence of catalase. The absorbancy at 280 m,u of I mg apoenzyme per ml was 1.4 for a r-em light path judging from biuret and micro-Kjeldahl assays. The spectrum of the apoenzyme sh owed an absorption peak at 280 m,u and ~ slight shoulder at about 290 m,u. The visible spect ru m had no absorption peak. The reconstituted holoenzyme showed absorption peaks at 375 mfJo and 455 m,u (Fig. 3). The addition of alanine to the holo enzyme under anaerobic conditions produced a bleaching of the visible colour . The sedimentation and elect ro phoret ic patterns showed homogeneous single

F ig . + Sedimentation patterns of the apoenzyme. Protein concentration, 0.7 %, 20 mM pyrophosphate buffer (pH 8.3) ; rotor speed, 51200 r e v.{min ; the photographs were taken 20, 40, 60 8o , 100 and 120 min after re achin g final speed; a verage t emperature 6° ; sed imentation is fr om r ight to left.

peak (Figs. 4 and 5). S020 .lV = 8.1 was obtained by extrapolation of the plots of the sedimentation constants against the apoenzyme concentrations to the ordinate. D0 20 ,w = 6.0 was also obtained by the same way. From these values the molecular

F ig. 5. E lectrophoresis of the a poenzyme in 20 mM p yrophosphate buffer (p H 8.3)· A 0.98 % solu t ion of the apoenzyme was used. The electrophoresis was carried out at 20° using 80 V, 5 rnA a nd 60 0 for the d iagonal slit. Photographs of the ascending patterns (ar r ow) were at 30 min (1), 60 m in (2), 90 min (3) and 120 min (4)'

Biocbim . Biopbys. Acta,

105

(1965) 86- 9 9

CRYS TALLI ZAT ION OF D-AMINO ACID OXIDASE APOENZYME

93

o o ( 1)

0. 2

t

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.,

0 OJ

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0 .1

(2 )

t-

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« 20 FA D l,uM)

Fig. 6. Titra ti on of FAD with t he apoenzyme solution in 0 . 1 M pyrophosphat e buffer (pH 8.3). Open circl es (1) sh ow t he ab so rb ancy after the addition of FAD t o 8.85 ,ti M of the apoenzyme so lution , and filled circles (2), the absorbancy a ft er reduction with nc-alanine.

weight of the apoenzyme was calculated as 125 000, assuming that the par tial speci fic volume of the apoenzyme was 0.74. FAD which could bind to the apoenzyme was also determin ed by dialysis equilibrium and titration of the apoenzyme solution with FAD (Fig. 6). Bot h result s showed that 16 mzzmoles of FAD were bound to I mg of apoenzy me. This corresponds to 2 moles of FAD per mole of apoenzyme . The effect of P CM B on the enzyme The changes in the absorbancy at

250

I (B)

(A)

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o o

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- -- - - - - - - - - - - -- - - - (1)' :2 4 6 Time (min)

8

10

0

0

10

Time (min)

F ig. 7. A : Increment in the a bso rbancy at 2jO mil on reaction of PCME wi th the enzymeIrr 0.1 M pyrophosphate buffer (p H 8.3). T he concentration of the apoenzyme was 3.7 fLM, an d the molar ratio of PCME t o th e apoenzyme added was I -I ' , 0.86; 2-2 ' , 5.17 ; 3-3'. 10.33 respectively Solid lines sho w t h e r eaction of PCME with t h e apoenzyme and broken lines the reaction in the presence of I51lM FAD, ad de d 30 m in before t he a dd ition of P CME . E : 0.13 mg of trypsin wa s added to 3.0 ml of the reaction solution for Curve 3 wh en the turbidity was no longer increasing

B iochim, B iophys . Acta,

I Oj

(1965) 86-99

Y. MIYAKE

94

et al.

of PCMB were followed. There were three phases, namely an initial rapid, a subsequent slow, and then a final marked increment in the absorbancy (Fig. 7A). During this last increment in the absorbancy, it was observed that the reaction mixture became turbid, but on addition of trypsin the solution became clear again (Fig. 7B). However, the last rapid increment was scarcely observed with the holoenzyme (Fig. 7A). From these observations, it seemed likely that the initial rapid increment is due to the interaction of the apoenzyme with PCMB, as already reported by BOYER 20, and the last marked increment to the denaturation of the apoenzyme by peMB, so that the initial interaction was almost completed within z or 3 min. Fig. 8 shows the effect of varying the ratio of PCMB to apoenzyme concentration on 0.3

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0

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.

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0.1

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Fig. 8. The effect of increase in PCME concentration on the reaction of PCME with the apoenzyme. The symbols . - . and . - . show the reaction in the presence and absence of FAD respectively. The concentration of the apoenzyme and FAD added were 3.7,uM and IS {I.M respectively. Readings for these two curves were taken 2 min after mixing. Open circles show the reaction of PCMB with apoenzyme digested by trypsin. o. I 3 mg of trypsin was added to the apoenzyme and digestion was carried out for 40 min at 30°, and the absorbancy was measured 3 h after the addition of peME.

the changes in the absorbancy at 250 mp" 2 min after mixing. The curves showed that the increment of the absorbancy was almost saturated at a molar ratio of about 3 to 4. In addition to the large increment, a gradual increment in the absorbancy was observed on increasing the ratio (curves were shown by filled squares and filled circles in Fig. 8). The details of this gradual increment are still obscure, but it seems reasonable that this was due to a slow interaction of the enzyme with PCMB or to the initial stage of denaturation. With holoenzyme the absorbancy increased very slowly with time, and was five times as large as the initial rapid increment after 24 h reaction at 5°, and no turbidity appeared during this period. This suggests the existence of SH groups in the enzyme which react slowly with PCME. The slope of the apoenzyme curve (filled circles in Fig. 8) was a little more than that of the holoenzyme. This is probably due to the initial stage of denaturation. From extrapolation of the slope to the ordinate and L1 emM-value of 6.81' mole-1 . crrr< at 250 mp, obtained for bound PCMB, the PCMB which reacted rapidly with the apoenzyme was calculated to be approx. 2 moles of PCMB per mole of apoenzyme. When enzyme solution was digested with trypsin before the reaction with peME, Biocbim; Biopbys. Acta, IOS (196S) 86-99

CRYSTALLIZATION OF D-AMINO ACID OXIDASE APOENZYME

95

a much larger increment in its absorbancy was observed and it was found that 10 moles of PCMB reacted with I mole of the apoenzyme (plots of open circles in Fig. 8). As shown in Fig. 7, it appears that the reaction hardly proceeded after the initial rapid increment of the absorbancy in the presence of FAD. This state was relatively stable within IS min, and the enzymic activity was reversible in the presence of GSH at 25° with a relatively high concentration of PCMB and excess of FAD. These results made it possible to analyse the kinetics of the mechanism of inhibition of the enzyme action by PCMB. Fig. 9 shows the effect of varying both the alanine

(3)

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200

400

I

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600

800

1/ [p - alanin!] (M)

Fig. g. Plot of reciprocal initial velocity of the enzyme action 1JS. reciprocal n-alanine concentration in the presence of various concentrations of peMB. The concentrations of peMB used were o !!M (I); I,95 !!M (2); 19.5 !!M (3). As the enzyme solution, 59 fIg of apoenzyme and 20 ftg of FAD were dissolved in O.I M pyrophosphate buffer (pH 8.3). Readings for the reaction rate were taken IO min after the addition of the substrate.

and PCMB concentrations on the reaction rate. The double-reciprocal plots of IJV versus I/[alalline] converged to the abscissa at every concentration of PCMB employed. The figure showed that the inhibition was apparently noncompetitive. This inhibition was shown to be partially noncompetitive from the following results. The inhibition constant (Kj ) was calculated from the following equation by the procedure of DIXON AND WEBB 2S : Kl = i[(H. - K p ' V 'R)/(K p ' V - H.)]

where i, Kg, K p , V and R represent the concentration of PCMB, dissociation constant of the enzyme-substrate complex, the slope of the plot of the reciprocal of the reaction rate vs. the reciprocal of the concentration of the substrate, the maximum velocity of the enzyme action, and the ratio of the rate constant of breakdown of the ternary complex (enzyme-substrate-PCMB) to that of enzyme-substrate complex respectively. The result is shown in Table II. The constant value of 3.6,uM was obtained, as shown in the second column of the table. This constant value could not have been obtained, if it was assumed that the ternary complex did not breakdown at all according to the equation K, = i[Ks/(Kp' V - K s)]. The results of this calBiochim, Biopbys. Acta, ras (1965) 86-99

96

Y. MIYAKE

et al.

TABLE II INHIBITION CONSTANT OF

FCMB

FOR TIlE ENZYME

Concn. K j (11M) from the equations ofPCMB ('1M) i(K./(](p· V - K.)) i[(K. - I
0.65 1.95 9·75

7. 2 12·9 23·3 30 • 2

19.5 0

Mean 3.6

culation are shown in the first column of the table. This was confirmed by an experiment on the effect of the concentration of PCMB on enzymic activity (Fig. ro), The enzymic activity did not disappear on increasing the concentration of PCMB, but attained a constant value of 56% of the activity of the native enzyme. With a high concentration of PCMB all the enzyme was considered to be converted to the form bound with PCMB.

100 0

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o

0

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50

0

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40

(PCMBJ /[opoenzymi'j Fig. 10. The effect of FeME on the initial velocity of the enzyme action. The experimental con drtions were the same as that shown in Fig. 9. The enzymic activity was measured manometrically at 25° after addition of 0.1 mmole of DL-alanine and various concentration of PCME. Abscissa represent the molar ratio of the concentration of PCME to the apoenzyme added.

The effect of Nagarse and urea on the enzyme The results that the adsorb ability on hydroxylapatite and the denaturative effect of PCMB on the enzyme differed in the presence and absence of FAD, suggested the difference in the nature of the protein moiety between the two states. The Nagarse digestion and urea denaturation were attempted to obtain information on Biocbim, Biopbys, Acta, 105 (1965) 86-99

CRYSTALLIZATION OF D-AMINO ACID OXIDASE APOENZYME 100

100

eo

80

97

~

c:

2o

c

60 :;;0

60

~"L>

0-

ilo

40 Eo

~ 40

."

....0

....o

o :;; 20

0

20 :;; c::"

"

c::

20 Time (min)

Fig. I I. Digestion of the apoenzyme and the holoenzyme by Nagarse. Open circles show the ratio of inactivation, filled circles the ratio of denaturation. The solid lines show the digestion of the holoenzyme and broken lines that of the apoenzyme.

this problem. As shown in Fig. II, the apoenzyme was easily digested by Nagarse, while the holoenzyme was strongly resistant to the digestion. On urea denaturation, the holoenzyme was more resistant than the apoenzyme, and it was also observed that the reduced enzyme was extremely stable for urea denaturation (Fig. I2). 100x

(3)

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~

t

.0 ~c

50

o '6

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ex:

0 0

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Fig. 12. The effect of urea on the apoenzyme and the holoenzyme in the oxidized and reduced states. Curve I, apoenzyme; Curve 2, oxidized enzyme; Curve 3, reduced enzyme.

DISCUSSION

As described in the early section of this paper, since the crystallization of this enzyme was achieved there have been many interesting reports on the mechanism of the enzyme action and the protein moiety. Among them, the evidence reported Biochim, Biopbys. Acta, 105 (1965) 86-99

98

Y. MIYAKE et al.

by YAGI et al? that the apoenzyme changes greatly in size and shape on addition of FAD attracted our attention. However, the purification of the apoenzyme described above, which was achieved under much milder conditions than acid and heat treatment, yielded an apparently homogeneous preparation, the sedimentation and diffusion constants obtained being almost the same as those of the holoenzyme rather than those of the apoenzyme described by YAGI et al ,', From these results, it appears that there is no apparent difference in the size and shape of the apoenzyme in the presence and absence of FAD. On the contrary. several results obtained suggested changes in the protein moiety: (r) the adsorbability on hydroxylapatite, (2) denaturative effect of PCME and urea, and (3) the susceptibility to Nagarse digestion of the enzyme differed in the presence and absence of FAD. In each case FAD protected the protein moiety from the effect of these treatments. It seems likely from these results that there is a limited conformational change in the protein moiety. Concerning the changes in the protein moiety, YAMANO et al.24 have already suggested that there are probably small changes in the active site. As CHARLWOOD 25 reported that the sedimentation constant of the enzyme at low concentrations decreased, one of the authors is now confirming this subject. The visible spectrum of the reconstituted holoenzyme differed from that observed by KUBO et al. 2 and MASSEY et al,», i.e. no distinct shoulder at about 490 mp was observed. As YAGI AND OZAWA 5 have already reported, this shoulder appeared on the addition of benzoic acid. However, the spectrum later reported by MASSEY AND GIBSON l 3 who obtained the holoenzyme without cleaving FAD from the apoenzyme, showed a very slight but distinct shoulder. Though the precise meaning of this difference is still unknow, the very slight modification might have occurred during the preparation of the apoenzyme. The observation that the crystalline apoenzyme showed a little lower specific activity than the holoenzyme, reported by MASSEY et ai», may be related to the difference in the spectrum described above. Concerning the SH groups in the enzyme it was found that there are two types of SH groups, with high and low reactivity, and it may be that the reactive SH groups in the enzyme are related to enzyme action directly or indirectly. From kinetic studies it seems that the reactive SH groups in the enzyme do not compete with the substrate. The role of SH groups in the binding of FAD, as reported by KUBO et al,2 and YAGI AND OZAWA 9 , could not be deduced from the present results. However, it is likely that FAD does not cOffiI?ete completely with PCMB. This was confirmed by fiuorimetric studies; the fluorescence of bound FAD was not increased by addition of PCMB within 3 min after the start of the reaction. Accordingly, the binding of PCMB to the enzyme may change the mode of binding of FAD or modify the structure of the active site, and this ternary complex may decrease the enzymic activity to 56% of that of the native enzyme. The discrepancy between the results of KUBO et al.2 and the present results on the number of detectable SH groups in the enzyme could be resolved by converting the absorbancy at 280 mp of r mg protein per ml from 1.0 to r.a, when the results showed fairly good agreement. ACKNOWLEDGMENT This work was in part supported by a research Grant from the Ministry of Education of Japan. Biocbim, Biopbys. Acta, lOS (I96S) 86-99

CRYSTALLIZATION OF V-AMINO ACID OXIDASE APOENZYME

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