d-amino acid oxidase i. dissociation and recombination of the holoenzyme

BIOCI-IIMICA, ET BIOPHYSICA ACTA

BBA 65162

D-AMINO ACID OXIDASE

I. DISSOCIATION AND RECOMBINATION OF T H E HOLOENZYME

357

MALCOLM DIXON AND K J E L L KLEPPE"

Department of Biochemistry, University of Cambridge, Cambridge (Great Britain) (Received October i3th, x964)

SUMMARY

I. The purified D-amino acid oxidase (D-amino acid:D2 oxidoreductase (deaminating), EC 1.4.3.3) of pig kidney has been studied by means of the 02 electrode, as a preliminary to a systematic study of its specificity.

2. This technique revealed changes in activity in the early stages after the addition of enzyme to the test solution; these are not observable by the usual manometric method. They are shown to be due to spontaneous dissociation of the holD- enzyme into FAD and apoenzyme on dilution.

3. The dissociation and association are not instantaneous, but require several minutes. The rate constant for the dissociation was found to be 0.45 miD -1, and that for the combination 1.8. lO 6 M -1 mid - : at 25 ° and pH 8.5. The rates are decreased when the enzyme combines with substrates or competitive inhibitors.

4. The equilibrium constant deduced from these values is 2.5" IO -~ M. The value calculated from equilibrium concentrations was 2.8. IO -~ M, in good agreement with previous values.

5- The rate of inhibition of the enzyme by p-chloromercuribenzoate is identical with the rate at which FAD dissociates from the enzyme, and is slowed to about the same extent by competitive inhibitors.

INTRODUCTION

Considerable advances have been made in the preparation of the flavoprotein enzyme D-amino acid oxidase (D-amino acid:O 2 oxidoreductase (deaminating), EC 1.4.3.3), and in the understanding of its mechanism of action, since the partial purification of the apoenzyme by NEGELEIN AND BR~MEL 1 in 1939. The holoenzyme was crystallized in 1958 by KUBO et al. ~ who stated that it contained about ten atoms of iron per r av in group. In 1961, however, MASSEY, PALMER AND BENNETT a also

* Present address: Norsk Hydro's Insti tute for Cancer Research, Oslo (Norwayl. Abbreviation: PCMB, p-chloromercuribenzoate.

Biochim. Biophys. Acta, 96 (1965) 357-367

358 M. DIXON, K. KLEPPE

crystallized the holoenzyme by a somewhat different procedure and showed that it was free from iron. YAGI et al.4, 5 have studied the physical properties of the enzyme and have concluded that it contains two molecules of FAD per molecule of protein.

The present study was undertaken as a preliminary to the extensive study of the specificity of the oxidase by means of the 02 electrode, described in the following paper s . Before this could be carried out it was necessary to account for certain changes in activity revealed by the electrode during the early stages of the reaction, but not observable by the usual manometric methods. With the latter methods readings cannot be obtained for several minutes after mixing the components of the test system owing to the necessity for temperature equilibration, whereas with the electrode there is no waiting period.

Experiments with the electrode showed that on mixing with the substrate the holoenzyme of D-amino acid oxidase, like that of L-amino acid oxidase (EC 1.4.3.2) is fully active at first, but that whereas the activity of the L enzyme remains constant, that of the D enzyme falls progressively, in the course o f a few minutes reaching a steady value which is only a fraction of the original. This inactivation is greatly retarded by the addition of various inhibitors, and is fully reversed by FAD, although not at once. The present paper describes experiments on this phenomenon.

MATERIALS

D-Alanine, the substrate, was obtained from Hoffmann-LaRoche & Co., FAD from the Sigma Chemical Co., 2-oxobutyric acid and PCMB from L. Light & Co., and the catalase (EC 1.11.1.6) was a stock solution of purified fiver catalase prepared in the laboratory.

Preparation of D-amino acid oxidase This enzyme was prepared from pig kidney according to the method of MASSEY,

PALMER AND BENNETT 3. After elution from the calcium phosphate--cellulose column, it was examined for homogeneity by starch-gel electrophoresis at pH 7.0. Only one protein band was observed, and this band contained all the D-amino acid oxidase activity.

The enzyme was also investigated by ultracentrifugation at pH 6.5 and 8.5, and in concentrations of 0.5 and 2.5 mg/ml. In all cases two peaks were observed, in agreement with the observations of other workers with the pure enzyme. This has been attributed to polymerization, but there is some evidence (see DISCUSSION) that it is due to a change in molecular shape rather than size. In our experiments the major component (s2o, w = 5.3 S) accounted for 90% and the minor one (s20,w---- 7.7 S) for only IO~/o of the total. In view of the results described below, these experiments were repeated in the presence of excess (6. lO -5 M) FAD, and also in the presence of 6.1o -5 M FAD and 1.O.lO -3 M 2-oxobutyrate, added 5 rain before placing in the ultracentrifuge cell. No changes in sedimentation coefficients were produced.

The method of preparation gives the fully active enzyme containing its full complement of ravin. The specific activity was found to agree, when allowance was made for the difference of temperature, with values obtained by others, and it was


D-AMINO ACID OXIDASE: DISSOCIATION AND COMBINATION 359

not increased by the addition of FAD. Spectrophotometric estimation of the rav in content gaw; a molecular weight per rav in group of 54 ooo.

A port.ion of the enzyme was crystallized, but this gave no advantage and for the work described here crystallization was omitted. In fact YAGI, OZAWA AND HA- RADA 7 have maintained that the crystals isolated by MASSEY'S procedure consist of a complex of the enzyme with benzoate, a competit ive inhibitor. In order to find out whether the concentration of benzoate in our enzyme preparation was large enough to affect the rate measurements, a small sample was freed from any traces that might have been present by means of the following procedure. The enzyme was first reduced by excess of D-alanine, then precipitated with ammonium sulphate and washed sew~ral times with 50% saturated ammonium sulphate, and finally dissolved in a small volume of o.o5 M phosphate buffer (pH 6.5) and dialysed against the same buffer overnight. The Km for D-alanine given by this sample was found to be identical with that given by the original enzyme, showing that any traces of benzoate in the preparation were too small to have any detectable effect on rate measurements.

METHODS

The measurements were carried out with the Clark 02 electrode in an apparatus somewhat similar to that described by PEEL 8. The cylindrical reaction vessel (2.8 cm long × 2.1 cm diameter) was immersed in a water bath set at 25 °, and rested on a small mag~aetic stirrer running at about 300 rev./min. A small glass-covered iron rod in the vessel gave continuous stirring of the contents. The electrode was fitted through a perspex disc which fitted the vessel bore very closely, and could be raised and lowered by a rack and pinion adjustment, The disc had a small hole through which additions could be made; this was closed by a plug during the measurements so tha t no oxygen could diffuse into or out of the vessel. No gas phase was present during the reaction and the height of the electrode was carefully adjusted to exclude all bubbles. A polarizing voltage of 0.6 V was applied to the electrode, and the current was passed through a sensitivity control to a Honeywell-Brown recorder with a chart speed of 0.5 in/min, using a chart with IOO equal divisions. The rate of utiliza- tion of the dissolved oxygen by the oxidase was thus given by the slope of the re- corded line.

Standard:ization of the electrode The majori ty of the experiments was carried out with the solutions initially

saturated with air. In this case the electrode was always standardized with distilled water salurated with air at 25 °. I t was found convenient to adjust the reading then given arbitrarily to 9 o on the chart by means of the sensitivity control. At standard barometric pressure, this reading therefore corresponded to an 02 content of 0.257/ZlItole/ml in the water 9, or I.O28 #moles 02 in the total volume of 4 ml normally used in the vessel. I t was verified that the reading was zero in the absence of 02 and that it was proportional to the 02 pressure over the whole range o - I arm. Thus the readings could be converted into /zmoles 02 in the vessel by multiplying by the constant 1.o28/9o = o.o114, this constant being analogous to the constant of a manometer.

The presence of dissolved substances, however, may reduce the aqueous

Biochim. Biophys. Acta, 96 (I965) 357-367


solubility of 02; in the present case, for example, the air-saturated reaction mixture contained appreciably less O 2 than the water, owing to the presence of the pyrophosphate buffer. The electrode nevertheless gives the same reading as with water, for it measures the activity rather than the concentration of the 02 (CHAPPELL10). I t therefore gives the same reading with all air-saturated aqueous solutions, whatever their O, content; thus the value of the constant may be changed by dissolved substances.

The effect is very easily measured as follows. 4 ml of air-saturated water at 25 ° containing a small amount of catalase are placed in the vessel. The electrode sensitivity is reduced to about half, and a small volume of H202 solution is added from a micro- syringe. The production of 02 causes an increase in the reading, which is complete in less than i min. The experiment is repeated with air-saturated pyrophosphate solution containing catalase, using exactly the same volume of H20~; the increase in the reading is now larger, since the O, solubility is reduced. The ratio of the two increases gives the correction to be applied to the constant. In the present case the presence of the pyrophosphate caused a difference of exactly IO%, so that the correct constant for use with pyrophosphate was O.OLO3. This does not, of course, affect the routine standardization, which is done by adjusting to 9 ° with air-saturated water, not pyrophosphate.

In the case of an enzyme with a reaction velocity which is dependent on the O~ concentration, it might be expected that a further correction would be necessary because the pyrophosphate, by reducing the 02 concentration, will also reduce the velocity of the enzyme reaction. This, however, is not the case, for the enzyme- catalysed reaction, like other chemical reactions, has a velocity which is determined by the activities, rather than the concentrations, of the reactants. The O 2 activity remains the same, so that the degree of saturation of the enzyme with Oo and the reaction velocity are unaffected.

In the routine tests the total volume was 4 ml. The reaction was started by adding the substrate (or in some experiments the enzyme) after a steady reading had been obtained, and the recording was allowed to proceed usually for 3 or 4 min. The curve was usually linear at least for the first 20 divisions, and the initial rate could readily be determined. The vessel was always screened from sunlight.

RESULTS

Nature of the reaction As no catalase was normally present in the tests, hydrogen peroxide accumu-

lated during the reaction:

R ' C H N H ~ ' C O O H + HaO + O 8.~- R ' C O ' C O O H + NH a + HaOz

I t has been stated xl that the hydrogen peroxide reacts with the oxo acid formed, but this is not the case under the conditions of these experiments. Fig. I shows the effect of addition of catalase after the reaction had proceeded for some distance, and it will be seen that the 02 then produced was exactly half the amount that had been con- sumed up to that point:

H20 ~ - H ~ O + ½03



showing that one molecule of peroxide was produced for each molecule of 02, and that this all :remained in solution until the catalase was added. After the addition the rate dropped to half its previous value, in agreement with the above equations. At these concentrations and time intervals the peroxide has no effect on the activity of the enzyme.

At alkaline pH values hydrogen peroxide does decompose to some extent, but the rate of decomposition is too small to affect the rate measurement in the region up to p H I L

1001 o-alanine, L

8(~-

60

==

4o

.~ catalase L)

2O

0 I I I I I I I I 0 4 8 12

Minutes

Fig. I. Progress of reac t ion and effect of add ing catalase . The reac t ion vessel con ta ined 20/~moles D-alanine, 12. 5/~g D-amino acid oxidase ho l oenzyme a n d 5-7" IO-2/ ,moles F A D in a to ta l v o l u m e of 4 ml o.a 5 M p y r o p h o s p h a t e buffer (pH 8.5). T e m p e r a t u r e 25 °. A t second arrow o. i ml of o.I % ca ta lase added.

Effect of dilution on the holoenzyme The stock solution of the holoenzyme was of course fairly concentrated (0. 5 mg

protein/Inl) and in this state could be stored for long periods without loss of activity, either in 0.05 M phosphate buffer (pH 6.5) (the usual method) or in 0.05 M pyrophosphate buffer (pH 8.5). In the activity test 0.025 ml of this solution was added to 4 ml of the test mixture, a dilution of 16o times. As seen in Fig. 2, Curve C, the enzyme i,~ at first fully active at this dilution without any added FAD. The reaction starts off rapidly, but whereas in Fig. I (where FAD was present) it continued for some t ime at this rate, here it falls off rapidly and finally settles down to a very slow rate. The obvious interpretation is that from the instant of dilution the enzyme begins tc dissociate into apoenzyme and FAD, after a time reaching an equilibrium state in which most of it is dissociated and inactive. In Curves A and B the enzyme



loo D-aMnine

FMN $

80 . . . . . . . .

60

~9

2G

I I I J I I I I 0 4 8 12

Minutes after adding alanine

Fig. 2. Progress curves after dilution of holoenzyme wi thout added FAD. For both curves A and B 12.5/~g holoenzyme had been left s tanding in 4 m] 0.05 M pyrophospha te buffer (pH 8.5) for a period of 12 min f rom the t ime of dilution, and 2o/tmoles D-alanine were then added at the first arrow. At the second arrow 5-7' IO-2/2moles FMN were added to A and 5.7' lO-2/~moles FAD to B. For Curve C the 12. 5 ~g holoenzyme from the stock solution were not added until the first arrow, when it was added s imultaneously with the alanine. The holoenzyme had been kept in stock solution of o. 5 mg/ml. Tempera ture 25 ° throughout .

was added 12 min before the D-alanine and was allowed to stand in the diluted state at 25 ° until by the time the D-alanine was added it had become almost inactive. Addition of FMN at the second arrow had no effect, but from the instant of addition of FAD, reformation of the holoenzyme begins, and soon the rate of the reaction becomeg practically equal to the initial rate in Curve C.

Fig. 3 shows this dilution effect more completely, giving the course of the inactivation from the moment of dilution in the presence of different amounts of total FAD. No FAD was added for the bottom curve, the 6. IO -s M FAD being the amount in the holoenzyme added. I t will be seen that added FAD has two effects: it reduces the net rate of the inactivation and it affects the final activity of the enzyme when the steady state is reached.

It is clear that the cause of the inactivation is the reversible dissociation of the holoenzyme into FAD and apoenzyme. Assuming that each binding site in the apoenzyme can combine with one molecule of FAD, and that the sites combine inde- pendently of one another, the equilibrium can be written

k+l E F ~ - E + F

k - 1

where E F represents the holoenzyme, E the apoenzyme (or more strictly a binding

Biochirn. Biophys. Acta, 96 (1965) 357-367


site therein) , and F s t ands for FAD. An a p p r o x i m a t e calculat ion of the ve loc i ty

cons tan t s 4+ 1 and k- l , and the equi l ibr ium cons tan t K, can be made f rom the curves. F r o m the ini t ia l t angen t to the lowest curve, k+ 1 = 0.45 min -1 approx ima te ly . The difference between this ra te and those of the o ther curves is due to the back react ion p roduced b y the a d d e d F A D , and from this difference in the case of Curves 2 and 3 k_l can be ca lcula ted to have a value of 1.8. lO 8 M -~ min -~.

,--,

7 .c E

E

.>_ "5

1.4 xl0-SM FAD

1.4 xiO-~ M FAD

7.0 x t0- 'M FAD

6.0x10 *M FAD (no FAD added)

I I I I I I 4 8 12

M i n u t e s af ter d i l u t i on

Fig. 3. Progress of inactivation of holoenzyme after dilution in presence of different concentrations of FAD. o.o25-ml samples of D-amino acid oxidase stock solution (0. 5 mg/ml) were added at time o to a total volume of 4 ml 0.05 M pyrophosphate buffer (pH 8.5) containing amounts of FAD sufficient to make up the total concentration shown. The activity was then measured at various times by the addition of 2o/*moles D-alanine.

The equi l ibr ium cons tan t can be o b t a i n e d e i t h e r from the ra t io of these r a t e cons tan t s or f rom the final act ivi t ies . Tak ing Curve 4, and expressing K as (E)(F)/ (EF), where (E), (F) and (EF) are the equi l ibr ium molar i t ies of free b inding sites, free F A D and the holoenzyme respect ively , K is easi ly ca lcu la ted to be 2.8. lO -7 M, whereas the ra t io k+l/k_ a = 2.5" lO -7 M. This is only s l ight ly higher t han values for K at 380 de te rmined b y o ther methodsaa, 14, which suppor t s the in te rp re ta t ion . Curves 2 and 3 give values abou t twice this, b u t i t is possible t ha t there m a y have been some compet i t ion f rom impur i t ies in the F A D a d d e d (cf. BURTON1*).

The ra tes of associat ion and dissociat ion are not g rea t ly affected b y p H in the region f rom 7 to IO, ne i ther are t hey dependen t (at p H 8.5) on the py rophospha t e concen t ra t ion .

Retardation by competitive inhibitors In tlhe presence of cer ta in compet i t ive inhibi tors such as 2 -oxobu ty ra t e the

ra tes of di,;sociation and associat ion are ve ry g rea t ly reduced; twenty- fo ld decreases



in the velocity constants for these processes are easily produced. The effect on the dissociation is illustrated in Fig. 4. In about 30 min the two curves come together and thereafter they remain coincident. In other words the effect is specifically on the rate, without any change in the equilibrium, and this implies that the rate constant of association k_ 1 must be reduced in the same proportion as k+ v The same result was obtained when benzoate was used instead of 2-oxobutyrate. It would appear from Fig. 2 that the substrate also has some effect in retarding the dissociation, and therefore one cannot calculate the rate constant from this figure.

100

80

>, 60 >

40

20

. . . . .

6.0 x 10-=M FADc~

0 ~ ' I I r I I I 4 8 12

Minutes after dilution

Fig. 4. Effect of an inhibitor on the rate of dissociation of the holoenzyme. 0.025 ml stock solution of D-amino acid oxidase holoenzyme diluted a t t ime o to a total volume of 4 ml in 0.o 5 M pyrophospha te buffer (pH 8.5) at 25 ° in the presence and absence of 5 ' lO-8 M 2-oxobutyra te (final concentration). The concentrat ion of FAD shown was derived f rom the holoenzyme.

Effects of sulfhydryl reagents on the holoemyme The effect of PCMB on the enzyme is illustrated in Fig. 5. Results of the

same type have also been obtained with other -SH reagents, such as iodoacetamide and chloroacetophenone. With no added FAD (lowest curve) the enzyme becomes inactivated at about the same rate in the presence of the -SH reagent as in its absence. The main difference is that now the activity falls to zero, and not to an equilibrium value. The effect of the reagent is much more evident in the presence of 1.4. lO-5 M FAD, which was sufficient in Fig. 3 to prevent inactivation but here only has a slight protective action against the reagent. The protective action of FAD has already been noted by FRISELL AND HELLERMAN 15.

The 2-oxobutyrate shows a marked protective action, even without any added FAD. This action might be produced in two ways: (a) by decreasing k+ 1 so that the FAD group is retained on the enzyme for a longer time or (b) by direct protection of a sulfhydryl group. On comparing Figs. 4 and 5 it can be seen that the rate of inactivation of the enzyme by the -SH reagent is about the same as the rate of disso-


D-AMINO ACID OXIDASE: DISSOCIATION AND COMBINATION

lOOm_ 'l

1.4 xlO'SM FAD ÷SxlO-=M 2-oxo-butyrate

80

365

60F-i \ o

40

20

10 20 30 Minutes after adding 6x lO -4 M PCMB

Fig. 5- Effect of FAD and inhibitor on the rate of inhibition of the holoenzyme by PCMB. Con- ditions as for Fig. 4, b u t PCMB added at t ime o in all cases to a concentrat ion of 6. lO .4 M, and act ivi ty measured at different t imes by adding 2o #moles D-alanine and 5.7" IO-=/zmoles FAD. For two curves FAD added to concentrat ion shown.

ciation of FAD in the presence of the inhibitor, and it would seem that the major protection provided by the latter is due to decreasing k+l, so that the real protector is the FAD group. The rate determining step is thus the loss of the FAD group, and as each molecule of apoenzyme is produced it is immediately at tacked by the reagent. With added FAD as well as the inhibitor, there is almost complete protection against the reagent.

DISCUSSION

Our understanding of the physical properties of flavoprotein catalysts is still far from complete. The basic problems in rav in catalysis are closely connected with the chemical properties of the flavins and the mode of their binding to the proteins, but at the moment too little is known about these factors.

The information available on the physical properties of D-amino acid oxidase is rather contradictory. MASSEY et al. 3 have postulated a molecular weight of 182 ooo with four FAD groups per molecule. CHARLWOOD et al. 16 have suggested that the enzyme is made up of units of molecular weight 45 7 °0 , its molecular weight depending upon the concentrations of total FAD and total protein ; but the evidence for this is insufficient, since it is based primarily on sedimentation data. YAGI et al. ¢ on the other hand have evidence that the molecular weight is 115 ooo with two FAD groups



per molecule, and also that, although the holoenzyme and its benzoate complex have sedimentation coefficients s20,w of 8.0 S and II.O S respectively, this is not due to any difference in molecular weight but to a large difference in the shape of the molecule. They suggest that such a change in shape may take place during catalysis, but since it involves drastic alterations in the helical structure of the molecule it seems more likely that it would occur over a fairly long period of time than that it would take part in the catalysis and be a mat ter of milliseconds. The oxidase can probably exist in a number of different conformations, all with catalytic activity.

The present work brings out the importance of always having excess of FAD present in kinetic work with D-amino acid oxidase, in order to avoid changes in activity during the measurements. This precaution has not always been observed in the past.

The binding of substances by enzymes may take place at widely different rates. The combination with substrates and inhibitors of course is normally very rapid. On the other hand, the reaction with metal ion activators such as Mn 2+, Co 2+ and Ni 2+ may be a mat ter of hours. With regard to the rate of binding of prosthetic groups by apoenzymes, apart from the present work we know of only one case in which measurements have been made, namely these on reduced NADP dehydrogenase (EC 1.6.99.1, the so-called 'old yellow enzyme') by THEORELL AND NYGAARD 17,18, using fluorescence methods. This flavoprotein, however, is infinitesimally dissociated compared with D-amino acid oxidase; moreover the dissociation is dependent on the action of certain particular anions, which is not the case for the oxidase. The velocity constant of dissociation of the oxidase is some IO ooo times greater than that for THEORELL'S enzyme; on the other hand the velocity constant of association is about 3 ° times greater for THEORELL'S enzyme than for the oxidase.

The retardation of both association and dissociation of the oxidase by competitive inhibitors (and probably by substrates also) is interesting. I t cannot be a combination between inhibitor and FAD-binding site, because if this were the case the inhibitor would compete with the FAD and alter the equilibrium constant, whereas K remains unchanged. Apparently the inhibitor combines with the substrate-binding site, and this is so close to the FAD site that it then obstructs the passage of the FAD both to and from its site.

The fact that when combined with FAD the enzyme is protected against - S H reagents, which otherwise rapidly at tack it, suggests that the bound FAD covers an essential - S H group, but that there is no such group in the substrate-binding site. The substrate may protect indirectly by preventing the FAD from leaving its site, so keeping the - S H group covered.

I t will be recalled that BURTON 1~ studied an inactivation of D-amino acid oxidase which has certain similarities with that described here, especially in the protective action of substrates and competitive inhibitors. The two processes are, however, quite distinct. The present process consists in the reversible conversion of the holoenzyme into the apoenzyme, whereas BURTON studied the irreversible inactivation of the apoenzyme.

Biochim. Biophys. Aura, 96 (1965) 357-367


ACKNOWLFDGEMENTS

We are grateful to the Royal Society for a grant to M. D. for the purchase of the 02 electrode outfit. One of us (K. K.) is indebted to the Norwegian Council for Scientific ~nd Industrial Research for the award of a Research Fellowship. We wish to thank Mr. P. KENWORTHY for valuable technical assistance.

R E F E R E N C E S

I E. NEGELEIN AND R. BRrMEL, Biochem. Z., 3o0 (1939) 225. 2 H. KUBO, M. YAMMANO, M. ITWASUBO, H. WATANI, T. SOYAMA, J. SHIRAISHI, S. SAWADA,

N. KAWASHIMA, S. MITANI AND K. ITO, Bull. Soc. Chim. Biol., 4 ° (1958) 28. 3 V. MASSEY, G. PALMER AND R. BENNETT, Biochim. Biophys. Acta, 48 (1961) I. 4 K. YAG], T. OZAWA AND T. Ooi, Biochim. Biophys. Acta, 54 (1961) 199. 5 K. YAG]: AND T. OZAWA, Biochim. Biophys. Acta, 81 (1964) 599- 6 M. DIXON AND K. KLEPPE, Biochim. Biophys. Acta, 96 (1965) 368. 7 K. YAG];, T. OZAWA AND M. HARADA, Nature, 188 (196o) 745. 8 J. L. P~EL, Biochem. J., 88 (1963) 296. 9 C. D. HODGMAN, Handbooh of Chemistry and Physics, Chemical Rubbe r , Cleveland, Ohio

(U.S.A.), 39th Ed. , 1957-8, p. 16o7. IO J. B. CE:APPELL, Biochem. J., 90 (1964) 225. I I A. MEISTER AND D. WELLNER, in P. O. BOYER, H. LARDY AND I~. MYRB.~.CK, The Enzymes,

Vol. 7, Academic Press, New York, 2nd Ed. , 1963, p. 61o. 12 K. BURTON, Biochem. J., 48 (1951) 458. 13 O. WARBURG AND W. CHRISTIAN, Biochem. Z., 298 (1938) 15o. 14 E. WALAAS AND O. WALAAS, Acta Chem. Scand., io (1956) 122. 15 W. R. FRISELL AND L. HELLERMAN, J. Biol. Chem., 225 (1957) 53. 16 P. A. CHARLWOOD, G. PALMER AND R. BENNETT, Biochim. Biophys. Acta, 5 ° (I96I) 17. 17 H. THEORELL AND A. P. NYGAARD, Acla Chem. Scand., 8 (1954) 877. 18 n . THEORELL AND A. P. NYGAARD, Acta Chem. Scand., 8 (1954) 1649.


d-amino acid oxidase i. dissociation and recombination of the holoenzyme

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