d-amino acid oxidase II. Specificity, competitive inhibition and reaction sequence

368

D-AMINO ACID OXIDASE

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 65163

II . SPECIFICITY, COMPETITIVE I N H I B I T I O N AND REACTION SEQUENCE

MALCOLM DIXON AND K JELL KLEPPE* Department of Biochemistry, University of Cambridge, Cambridge (Great Britain) (Received October I3th, 1964)

SUMMARY

I. A systematic study has been made of the specificity of purified D-amino acid oxidase (D-amino acid:02 oxidoreductase (deaminating), EC 1.4.3.3) of pig kidney, with measurements of V and Km for each amino acid substrate.

2. The enzyme has a high degree of specificity for 0 2 as hydrogen acceptor; a slight reaction occurs with certain dyes, but none with ferricyanide, cytochrome c, quinones, phenazine methosulphate or NAD.

3- In the activated state the D-amino acids show very large differences in reactivity, quite apart from any differences in affinity for the enzyme. The substrate with by far the highest value of V is D-proline. Among the other substrates the most reactive are D-methionine, D-alanine, D-norleucine, D-isoleucine and D-phenylalanine. Acidic and basic amino acids do not react, with the exception of D-ornithine. The weakly basic D-histidine reacts, as do all ring-containing D-amino acids. The enzyme is completely D specific.

4" The enzyme is competitively inhibited by straight-chain fa t ty acids. For these the value of I/Ki (the affinity) rises to a maximum with 5 C-atoms, thereafter falling off with increased chain-length. This parallels the effect of chain-length on I/Km for the corresponding aliphatic amino acid substrates, which suggests that I/Km gives an indication of substrate affinity.

5. The amines of the amino acids and most of the L-amino acids fail to inhibit ; 2-oxo acids and 2-hydroxy acids are stronger inhibitors than fa t ty acids. I t may be inferred that the carboxyl group is essential for combination but the amino group is not, that a suitable aliphatic group or ring makes a considerable contribution to the affinity, and that an amino group in the wrong configuration has a negative affinity which may overcome the positive affinities of the other groups.

6. The effect of varying O 2 concentration supports a reaction sequence in which the product dissociates from the enzyme before the reaction with 02 takes place.

" Present address: Norsk Hydro's Institute for Cancer Research. Oslo (Norway).

Biochim. Biophys. Acta, 96 (1965) 368-382

D-AMINO ACID OXIDASE: SPECIFICITY" 369

INTRODUCTION

The study of the specificity of enzymes plays an important part in the elucida- tion of reaction mechanisms. There are two aspects of specificity, however, namely the relation between the chemical structure of the substrate molecule and its ability to combine with the active centre of the enzyme, and the relation between the chemical structure of the substrate and its rate of reaction when combined with the active centre. For the study of reaction mechanisms the two effects of substrate structure should be clearly dissociated. If the reaction rates of different substrates are simply compared at a single arbitrary concentration, as is too often the case, it is not possible to tell whether the differences are due to differences of enzyme-substrate affinity or to differences in reactivity of the enzyme-substrate complexes.

I t is therefore necessary to test each substrate at a series of concentrations and by the st~mdard methods to determine in each case the value of V (the maximum velocity, obtained when the enzyme is saturated with substrate) and Km (the substrate concentration at which the velocity is half V). The value of V, unlike that of v (the velocity with an arbitrary substrate concentration), gives the rate of reaction of the activated substrate when effects due to the influence of affinity have been eliminated.

The Michaelis constant Kin, on the other hand, may throw valuable light on the specificity of combination with the active centre. I/Kin is often equal to the affinity, although in other cases, where V is relatively high, it gives only a minimum value for the affinity. The measurement of the inhibitor constant Kt for a series of inhibitors that compete with the substrate for the active centre may actually be even more valuable, for they are free from this ambiguity, and I/Ki gives the true affinity.

A s~cond requirement in valid specificity studies is that they should be carried out with highly purified enzymes; if not, it is always possible that some of the actions may be due to other enzymes present as impurities. It is unfortunately true that the number of published investigations in which both requirements were ful- filled is very small indeed; of these, probably the most extensive is the carboxylesterase ~;pecificity study by WEBB 1.

Among flavoenzymes, D-amino acid oxidase (D-amino acid:O.2 oxidoreductase (deaminating), EC 1.4.3.3) is partiqularly suitable for specificity studies and it has already been investigated from this point of view by a number of workers 2-5. In these studies, however, the two requirements mentioned above have unfortunately not been met. Much of the work has been done with comparatively crude enzyme preparations, and V and Km have not been determined for the various substrates. Since the holoenzyme can now be prepared in the pure form ~, it was decided to carry out a systematic study of its specificity, measuring these quantities for each substrate. In addition to this the acceptor specificity, affinities of competitive inhibitors, and effect of O~ concentration have been studied. Some attempt has been made to assess the contributions of the different parts of the molecule

NH2 I

R--CH--COOH


37 ° M. DIXON, K. KLEPPE

to the power of combination with the enzyme and the dependence of the reactivity on the nature of the R group, and to get some idea of the nature and action of the active centre.

MATERIALS

The oxidase preparation was the same as that used for the preceding paper ~. The FAD was obtained from the Sigma Chemical Co. The majori ty of the D-amino aCids were products of L. Light & Co. ; they were checked for impurities by paper electrophoresis and paper chromatography, and they were also tested for absence of the z forms with crystalline snake venom L-amino acid oxidase (EC 1.4.3.2 ) (Sigma). Glycine was recrystalhzed from Analar grade glycine in 30% ethanol, and the purity checked by analysing a sample in a Technicon amino acid analyser, which showed that it was quite free from other amino acids. The peptides were from the Nutritional Biochemical Corporation of U.S.A., and most of the L-amino acids and the fa t ty acids were from the British Drug Houses Ltd. D-2-Hydroxy-3-methyl-n- valeric acid, DL-2-hydroxy-n-butyric acid and DL-2-hydroxy-n-octanoic acid were gifts from Dr P. K. TVBBS of this Department.

METHODS

A cceptor specificity The reduction of the various acceptors was followed in a Beckman recording

spectrophotometer by the increase or decrease in absorbance at the appropriate wavelengths. The reactions were carried out at 25 ° under anaerobic conditions, using a I -cm spectrophotometric cell fused to the top part of a Thunberg tube, so that it could be evacuated. FAD was added to prevent any loss of activity by dissociation of the enzyme. V and Km were obtained by Lineweaver-Burk plots.

Substrate specificity and competitive inhibition The 02 electrode equipment was used as described in the preceding paper 7.

The standard conditions were as follows: 0.025 ml enzyme (stock solution of 0.5 rag/ ml), o.I ml 5.7"1o-4 M FAD, varying amounts of substrate or inhibitor, 0.05 M pyrophosphate buffer (pH 8.5) to a total volume of 4 ml at a temperature of 25 °.

For each substrate the rate was measured at 6-1o different concentrations and a Lineweaver-Burk plot made for each, straight lines being obtained in all cases. In many cases plots were obtained with several different enzyme preparations, and these were always found to give the same values of Km and of V (relative to D- alanine). In order to allow for any slight differences of activity between the different oxidase preparations, a parallel determination was always done with D-alanine and the value of V for each substrate was expressed in relation to that for alanine, taken as IOO. For each inhibitor the value of Ki was determined by the graphical method of DIXON 8.

Throughout the specificity and inhibition studies the 02 concentration used was that of the air-saturated buffer solution, but for studying the effect of 02 concentration on the rate of oxidation the solutions were saturated with oxygen or nitrogen from cylinders to give the desired concentration.


D-AMIN O ACID O X I D A S E : S P E C I F I C I T Y 371

RESULTS

A cceptor specificity The results of this s tudy given in Table I clearly show that D-amino acid

oxidase is very specific for oxygen as acceptor, at any rate under these conditions. Of the other acceptors tried, only the indophenol and methylene blue showed any detectable reaction, and these at a maximum rate of less than one-hundredth of the oxygen rate. I t is rather surprising that neither ferricyanide nor phenazine methosulphate react at all, as these are generally considered to be good acceptors for flavoproteins; the behaviour with phenazine is in marked contrast with that of L- amino acid oxidase, which was found to react very rapidly with it, as already found by other workers. I t was verified that the negative result with ferricyanide was not due to inactivation of the enzyme.

T A B L E I

ACCEPTOR SPECIFICITY OF D-AMINO ACID OXIDASE

T e s t c o n d i t i o n s : o . I m l 0.2 M D-a lan ine , o . I m l 5 . 7 . i o - 4 M F A D , o.o25 m l d i l u t e d e n z y m e , a c c e p t o r c o n c e n t r a t i o n v a r i e d , 0 .05 M p y r o p h o s p h a t e b u f f e r ( p H 8.5) to a t o t a l v o l u m e o f 4 ml . T e m p e r a t u r e 25 °. D-Ala n ine c o n c e n t r a t i o n 5" IO-a M. K m is h e r e t h e Micbae l i s c o n s t a n t fo r t h e a c c e p t o r , n o t fo r t h e s u b s t r a t e .

A cceptor V Km (Izmoleslminl (tiM) mg)

F e r r i c y a n i d e o - - C y t o c h r o m e c o - - M e n a d i o n e o - - Q u i n o n e o - - F M N o - - R i b o f l a v i n o - - P h e n a z i n e m e t h o s u l p h a t e o - - N A D o - - M e t h y l e n e b l u e o.o 4 6 7 2 , 6 - D i c h l o r o p h e n o l i n d o p h e n o l o .o9 15 O x y g e n 12.o T 8o

The,;e results were all obtained with D-alanine as substrate. Other substrates may well ~ v e different values for Km and V for these acceptors, but it is unlikely that the overall picture would change much.

The question why some flavoproteins react with oxygen and some do not is one of the most fundamental unsolved problems in the field of flavoprotein catalysis, and much more information is needed about the effect on the behaviour of FAD of the nature of its binding to proteins.

Substrate ,.~pecificity The results of the substrate specificity study are shown in Fig. I, a and b.

The D-amino acids have been arranged in groups according to the nature of R. At the left is shown the structure of R, to which the group - - C H N H , . COOH must be deemed to be attached, and the corresponding values of the maximum velocity V

Biochim. Biophys. Aaa , 96 (1965) 3 6 8 -3 8 2

V 50 100 0

R

H-

CH 3-

C H3.CH 2 -

CH3.CH2.CH 2-

CH3.CH2.CH2.CH 2-

DL- CH3.(CH2)s-

(CH3)2:'

(CH3)2.CH -

(CH3)2.CH.CH2 -

CH3.CH2.CH (CH3)-

CH2OH-

CH3.CHOH-

CH3.S.CH2.CH 2 -

C H3.CH2.S.CH2.C H2-

DL- CH2SH.CH2-

(CH3)2.CSH-

/CH 2- S".C H 2 _

m m

m

I I I

/K m (raM) -I 5 10 15

I i

372 M. DIXON, K. KLEPPE

Fig. I. a. Substrate specif icity of D-amino acid oxidase at p H 8. 5. V represents the m a x i m u m ve loc i ty w i t h excess substrate in relat ion to t h a t for n-alanine, t aken as IOO. A circle denotes zero velocity .

and of I/Km are shown in the form of horizontal histograms. V is given in relation to V for D-alanine, taken as ioo. Where V is zero this is indicated by a circle on the zero line; in such cases of course Km cannot be determined. V represents the relative reactivity, and I/Km is closely connected with, and in many cases probably equal to, the affinity.

This diagram shows the situation for air and pH 8.5, and under other conditions its appearance might be modified to some extent. Both V and Km show some dependence on the oxygen concentration, but not to the same extent for different amino acids, so that with pure oxygen there would be some relative differences. Moreover it is shown in the following paper that the pH curves for different amino acids are by no means the same. For example V for the first amino acid, glycine, increases up to pH IO, while that for D-methionine decreases, so that at pH IO, V is actually higher for glycine than for methionine.

The existence of a separate glycine oxidase was postulated in 1944 by RATNER, NOClTO AND GREEN9; however, they stated that their preparation still contained D- amino acid oxidase, and more recently some doubts on the subject have been expressed TM. Glycine certainly has a considerable reactivity with our purified ])-amino acid oxidase preparation, and we have been unable to separate the glycine oxidase


D-AMINO ACID OXIDASE : SPECIFICITY

R O

COOH.CH2-,

COOH CH:2 CH2-

DL- CH2NH2.CH 2.CH2-

DL- CH 2N H2.('CH2)3-

CONH2.CH2-

~ -CH2-

DL- H o H - ~ - CH2 -

( ~ C - C H 2 -

k~V/~N/C H H

H C = C - C H 2- q L N~C,,-N H

H

H2C--CH 2 DL- ~ I

H2C.,N/CH.CO OH H

50 100 I I

l//Kin (raM) "t

5 10 15 I I I

I

--~ 30~

373

Fig. I.b. Substrate specif icity of D-amino acid oxidase at p H 8.5. V represents the m a x i m u m ve loc i ty wi th excess substrate in relation to that for D-alanine, taken as ioo. A circle denotes zero ve loc i ty .

activity :From the D-amino acid oxidase activity by electrophoresis in starch gel. Moreover the Km obtained for glycine in this work is of the same order of magnitude as that obtained by RATNER et al. 9.

In the straight-chain aliphatic series the value of I/Kin rises regularly to a maximum with D-norvaline and then falls. This appears to be a true effect of the chain-length of the R-group on the affinity; the large difference between amino- butyric acid and norvaline, for instance, must be due to this cause, since they have about the same value of V, and the fact that the same R-groups produce similar changes !in affinity in inhibitors also provides strong evidence. The value of V, unlike that of I/Kin, rises to a maximum with D-alanine, then falls, rises to a second maximum with D-norleucine, and finally falls to zero.

In the branched-chain series there is a somewhat similar situation, with a maximum in I/Km again with a chain-length of 3 C atoms (not counting the methyl group) and a dip in V with a second maximum at 4 C atoms. The first member of the series, D-amino isobutyric acid, could not be expected to react, having no a- hydrogen atom.

The hydroxy-amino acids have moderate velocities, with very low values of I/Km. The sulphur-containing amino acids give higher values both of V and of I/Kin; D--methionine in fact is even more reactive than D-alanine. An interesting case is 3-mercapto-D-valine, which does not react at all, and this is also true of lanthionine.

Biochim. Biophys. Acta, 96 (I965) 368-382


None of the dicarboxylic acids or the diamino acids react at all, with the exception of ornithine; lysine, however, seems to react slowly at higher pH values.

All the ring-containing amino acids are quite reactive, with rather small I l K m values. D-Proline is outstanding, with a maximum velocity about three times as great as any other amino acid.

The strict D specificity of the enzyme was confirmed, so far as reactivity is concerned; no reaction at all could be detected with any L-amino acid, even in high concentration. On the other hand the D specificity is not so strict with regard to combining power, and it will be shown that certain R-groups confer some power of combining with the active centre even on L-amino acids.

Looking at the general picture it might be thought that there is an inverse relationship between V and I/Km, so that when one is high the other is low. But this is certainly not a general rule ; there are many cases in which one varies without the other (compare for example threonine with cysteine, or leucine with histidine), or where they vary in the same direction (compare glycine with alanine, serine with isoleucine, methionine with threonine, phenylalanine with dihydroxyphenylalanine, or t ryptophan with histidine). Thus it cannot be concluded that any inverse relationship there may be is due to the contribution of the same rate constant k+ 3 to the ex- pressions for V and Km (see the equations given later).

Inhibitor specificity I t has been known for some time that certain aromatic acids are good inhibitors

of D-amino acid oxidase n. We find that long-chain saturated afiphatic acids are also good competitive inhibitors of the enzyme, and the results obtained with these and with 2-hydroxy and 2-keto acids are shown in Figs. 2 and 3 and Table II.

T A B L E I I

I N H I B I T I O N OF O X I D A T I O N OF D - A L A N I N E B Y S O D I U M SALTS OF 2 - H Y D R O X Y A N D 2 - K E T O ACIDS

Test condi t ions as for Table L

Inhibitor Inhibition by o.oz M (%)

D-Malate o D-Lacta te 22 DL- 2- H y d r o x y b u t y r a t e 9 I D-2 -Hydroxy -3 -me thy lva l e r a t e 63 DL-2-Hydroxyoc tanoa te 77 P y r u v a t e I I 2 - O x o b u t y r a t e 86

Fig. 2 shows with lauric acid that the inhibition is purely competitive with the amino acid substrate. The lower part of Fig. 3 shows the affinity (I/Kt) of the series of fa t ty acids for the enzyme in relation to the total number of C atoms in their molecules. The upper part of the same figure shows I /Km for the corresponding amino acids, and the two series are aligned so that the same R-groups in the R- -CH2- -COOH and the R - - C H N H 2 - - C O O H molecules are together. The close correspondence between the effect of chain-length of the R-group on the affinity in


D-AMINO ACID OXIDASE :SPECIFICITY

J 1,6L

375

E

E

"5

0.4

0 0.4 0.8 1.2 1.6 2.0

1/[o-alanine] (mM) "~

Fig. 2. A Lineweaver-Burk plot showing the competitive nature of the inhibition of D-amino acid oxidase by fatty acids.

the case of the inhibitors and on I/Kin for the amino acids is strong evidence that the latter effect is a genuine effect on amino acid affinity. I t is evident that the R-group makes a l~.rge contribution to the affinity, that the enzyme has a considerable affinity for aliphatic groups, that each - - C H 2 - group adds its own contribution to the affÉnity, but that when a certain point has been reached the addition of one more - - C H , - - group produces a marked decrease.

Several other series of related compounds were tested for competitive inhibition in a semi-quantitative way, namely by determining the percentage inhibition produced by a concentration double that of the substrate, but without carrying out complete determinations of KI, which would have required a very large amount of work. Table I I shows the inhibition produced by 2-hydroxy and 2-keto acids. Again the repelling effect of a second carboxyl group is seen with D-malate, preventing it from combining with the enzyme, but all the others inhibit, to an extent not very greatly different from the fa t ty acids. With increasing chain-length there is again a rise followed by a fall, and the hydroxy and the keto acids seem to be about equally effective. I t is evident from all these results that the a-amino group is not essential for combination with the active centre, and that it makes no maior contribution to the affinity.

On the other hand it is clear from the results in Table I I I that an a-amino group in the wrong (L) configuration makes a negative contribution to the affinity



12 7

8 E

3

E 2

S

O0 2

D-amino-ac ids

II

Fatty ac ids

I I

i I , , 4 6 8

C atoms in molecule

II 10 12

Fig. 3. Effect of chain-length of f a t ty acid inhibitors on their affinity for the enzyme, compared wi th the corresponding D-amino acids. Kt determined wi th D-alanine as substrate .

TABLE III

INHIBITION OF OXIDATION OF D-ALANINE BY L-AMINO ACIDS

Test conditions as for Table I.

Inhibitor Inhibition by o.oz M (%)

Glycine o L-Alanine o L-Valine o L-Norvaline 75 L-Leucine 35 L-Isoleucine o L-Cysteine o L-Methionine 4 ° L-Serine o L-Threonine o L-Aspartate o L-Glutamate o L-Arginine o L-Ornithine o L-Lysine o L-Tryptophan o L-Phenylalanine o L-Histidine o L-Proline o


D-AMINO ACID OXlDASE :SPECIFICITY 377

sufficient ~o overcome all but the strongest positive contributions of the R-group such as occur in norvaline and leucine (compare Fig. Ia), though admittedly one would have expected L-cysteine to inhibit rather than L-methionine.

In contrast to the a-amino group, the carboxyl group is very important for combination, and its removal (even though the amino group is left) abolishes the ability to compete for the active centre, except weakly in two cases. This is shown by Table ]IV.

T A B L E IV

I N H I B I T I O N OF O X I D A T I O N OF D - A L A N I N E B Y A M I N E S


Inhibitor Inhibition by o.oi U (%)

Methylamine o Ethylamine o n-Buty lamine o n-Hexylamine o Cyclohexylamine o Pyrrolidine 12 Aniline 2 z A m m o n i u m chloride o

Fin:~lly the attachment of an amino acid residue to the amino group, although it abolishes all reactivity, does not in all cases abolish the power of combining with the enzyme in competition with the substrate, as shown in Table V.

T A B L E V

I N H I B I T I O N OF O X I D A T I O N OF D-ALANINE BY PEPTIDES


Inhibitor Inhibition by o.or M (%)

Glycyl-glycine o Glycyl-glycyl-glycine o Glycyl-glycyl-glycyl-glycine o DL-Leucyl-glycine 2 Glycyl-DL- valine i o GlycyI-DL- norvaline 13 GlycyI-DL- norleucine 9 Glycyl-DL-methionine o Glyc yl-DL- ethionine 3 Glycyl-DL-tryptophan 52 Glycyl-DL- phenylalanine 2 i Histidyl-histidine 22

Effect of oxygen concentration The results of MASSEY et al.e, a2 have been confirmed and extended. Fig. 4

shows a Lineweaver-Burk plot for D-alanine at different O, concentrations; parallel



0.6]

E

'~, 0.4 ).~

,>,.%,

0.2

0.4 0.8 ,.2 ,.6 2 .o 1/[D-alanine~ (raM)"

Fig. 4. L i n e w e a v e r - B u r k plots for D-alanine wi th different O 3 concent ra t ions .

lines are obtained, which means that a change of 02 concentration has the same effect on V as it has on Kin.

These and similar results throw some light on the sequence of steps in the oxidase reaction. There are two main possibilities. In one the oxidation product of the substrate leaves the reduced enzyme before the reaction with oxygen takes place; in the other the product remains combined with the enzyme until after the reaction with oxygen, and leaves the oxidized enzyme. The sequences may be written thus:

k+l k+o. k+ a k+ 4 E + S .~- E S ~ E ' P ~- E" + P, E ' + 0, 2 - - + E + H20, .,

k - , k - 2 k - s

k+t k+ 2 k+ 5 k+n E + S ,~- E S ~ E 'P , E ' P + 0 3--->EP + H20 v E P ~ - E + P

k - 1 k l k - e

(I)

(1i)

where E ' represents a reduced form of the enzyme. The application of steady-state kinetics makes it possible to decide experimentally between these two mechanisms.

We continue to write V and v for the velocity when the substrate concentration is, or is not, effectively infinite. Km is, as before, the Michaelis constant for the substrate, and K 0 will be written for the Michaelis constant for 03. The superscript 0 will denote infinite 03 concentration; thus V ° represents the velocity when both substrate and 03 concentrations are infinite, and v ° the velocity when the O2 concentration is infinite but the substrate concentration is not. Km° is the Michaelis constant for the substrate with infinite 02 concentration, and K0s will be used for the Michaelis constant for 03 with infinite substrate concentration. The four constants Kin, Ko, Km° and K0s can be determined experimentally from plots of I/V against I/IS] or I/[02~.

Bioch,m. Biophys. Acta, 96 (1965) 368-382

D - A M I N O A C I D O X I D A S E : S P E C I F I C I T Y 379

Application of the equation

V o V

K m ° Ko s I + - - - +

L s ] [0,]

where k+2k+se

V o = k+ 2 + k-~ + k+3

k-lk-~ + k-xk+8 + k+~k+a K m o =

k+lk+2 + k+lk-z + k+xk+3

kink+8 KoS

k+2k+4 + k-,k+4 + k + s h + i

steady-state treatment to Mechanism I gives the rate

The other quantities are related to these as follows

V o V 0 K m o Kos V v ° - - - K m K 0 - - - -

Kos Km° KoS K m ° I + - - I + - - I + - - i + - -

[02] [s] [o~] Is]

The. reason for the results of Fig. 4 wiU now become clear. The intercepts of the lines are determined by Km and V, and since an alteration of 0 2 concentration will affect: both in the same way the lines will remain parallel.

This is not the case for Mechanism II. This yields the rate equation

V

V o

K m ° /~o s /~so

i E-ST+ [s] [o,-----]

where the constants are now represented by different combinations of rate constants, which it is unnecessary to give. In this case while V is still given by V°/(I + Kos / [O21), Km is given by (Kin ° + Ks0/[O21)/(I + Kos/[O2~- ), so that the intercepts will be differently affected by a change in 02 concentrati~i~ and the lines will not remain parallel. This supports Mechanism I as against Mec~lanism II.

Further evidence is provided by a compariso n of different amino acids. Reci- procal plots were obtained in terms of both substrate and 02 for seven amino acids, and the values of the four K's determined from them. These are shown in Table VI. It will be clear from the above equations for Mechanism I that for any substrate concentration if v ° is divided by K o the result is equal to V°/Ko s and further that this is equal to k+4e. As k+ 4 is the rate constant for the oxidation of the reduced enzyme by 0 2 , it will be independent of the nature of the amino acid, and if the velocity is expressed per mg of enzyme, e may be taken as the same for all. Thus if Mechanism I is correct, k+4e and therefore v°/Ko will be the same for all amino acids. Table VI shows that although the other constants vary widely from one amino acid to another, this quantity remains approximately constant. This is strong evidence in favour of Mechanism I. Calculating the enzyme concentration in terms of its ravin groups, i.e. taking i mg as 1.85' I0 -s moles, the value of k+4 at 25 ° would be about 4.2" lO s min -1 M -1.

Biochim. Biophys. Acta, 9 6 ( I 9 6 5 ) 3 6 8 - 3 8 2

3 8 0 M. DIXON, K. KLEPPE

T A B L E VI

KINETIC CONSTANTS OF D-AMINO ACID

All K ' s expressed in #M, v ° in /*moles subs t r a t e .

OXIDASE

nlin -1 mg -1. Km de t e rmined wi th air, K o and v ° w i t h 5 mM

Substrate Kra K ~ K o Ko-q v ° v°/Ko

D-Alanine 18oo 4 i o o 18o 33 ° 12.2 o.o68 D-2-Aminobu ty ra te 46o 54 ° 41 46 3.6 o.o88 n -Norva l ine 63 71 29 3 ° 2. 5 o.o86 D-Norleucine 400 640 14o 16o 11.8 o.085 D-Valine 14oo 29o0 17o 270 lO.8 o.065 D-Methionine 83o 39 oo 53 ° 94 ° 33.5 o.o63 D-Proline 17oo 96oo 420 i34o 37.o 0.088

Mechanism I I gives for the quotient a complex expression, involving five rate constants and the substrate concentration, which would certainly give different values with different amino acids. Further evidence against this mechanism comes from the fact that under anaerobic conditions the oxidation of D-amino acids is reversible, that is to say the formation of D-amino acids from keto acids and am- monia can be demonstrated in the absence of O v provided that another D-amino acid is present to reduce the enzyme 13. This would seem to be incompatible with a mechanism in which the step involving 0 2 must precede the liberation of product.

I t should be noted that the symbol E ' is not intended to imply that the enzyme is fully reduced; in view of the work of MASSEY ~,1~ and others 14 it is more likely to be another form in wkich the rav in is only partially reduced.

DISCUSSION

The study of D-amino acid oxidase specificity has given interesting results both in relation to the acceptor and to the substrate. The enzyme is surprisingly specific for 02 as acceptor;i even phenazine methosulphate is inactive, in marked contrast to its extremely rapid reaction with L-amino acid oxidase under the same conditions. In view of the ease with which ravin nucleotides in the free state are oxidized by a variety of acceptors, the high degree of acceptor specificity of flavoproteins is remarkable, and merits further study. There is no known reason why some react with 02 and others do not. Some, but not others, react with ferricyanide, or with quinones, or with phenazine methosulphate, or with cytochrome c. I t is probably true to say that there is no single acceptor which will react with all the known flavoproteins. The enzyme protein clearly must have a profound influence on the reactivity of the rav in bound to it. The reactivity of this enzyme towards 03 cannot be due to an iron atom, as it has been freed from all iron.

With regard to the specificity of the enzyme-substrate combination, the results have shown that the active centre has a strong affinity, apart from the essential carboxyl group, for an alkyl group of moderate chain-length, so that the substrate must be held largely by Van der Waals forces. This alkyl-binding site, however, is evidently of limited extent and unable to accommodate a chain of more


D-AMINO ACID OXIDASE :SPECIFICITY 381

than 3 or 4 C atoms, since any increase of chain-length causes a marked drop in affinity. A ring can be accommodated. Little contribution to the affinity is made by the part of the substrate molecule that reacts, namely the a-amino group. Perhaps in the D position it stands away from the enzyme surface, so that it neither hinders nor helps combination; in the L position, on the contrary, it definitely hinders combination, although a few L-amino acids with specially strongly attracting alkyl groups may be drawn to the enzymes through these groups in spite of the hindrance. The enzyme, however, is strictly n specific, and even when combined these L-amino acids fail to react.

Few n-amino acids fail to react. These have either an alkyl group with too long a chain, or a charged group (particularly a negative group) within the R-group. Hydroxy acids are not oxidized, although it has been stated that they are oxidized by the L-amino acid oxidase of mammalian tissues 15.

Very few similar studies on other enzymes are available for comparison, i.e. studies on pure enzymes in which V and Km have been measured. The most systematic of these is that of WEBB 1 on carboxylesterase (EC 3.I . I . I ) , and it is interesting to note that there are certain similarities. Here also the binding power of the part of the substrate molecule which reacts is less than that of the alkyl groups. With increase of chain-length of the alkyl group there is an increase of affinity to a maximum, in this case with 4-6 C atoms with a fall on further increase of chain-length. The alkyl-binding site can also bind rings. An obvious difference is that no free carboxyl group is needed in the substrate. WEBB was able to form a tentative picture of the active centre on the basis of his results.

The picture at which we arrive for the active centre of D-amino acid oxidase is somewhat as follows. At one end is a positively charged group which combines with the - - C O O - group in the substrate. At the other end is a hydrophobic site binding the alkyl group, each - - C H ~ - - of which adds a contribution to the affinity; this site can accommodate a ring (possibly on edge), but is of limited extent perhaps because of some polar group or bulky obstruction beyond it, so that it can only accommodate up to 4 C atoms. The a-C atom of the substrate lies between, probably with its two C-C bonds parallel with the onzyme surface, so that when the D-amino group is c~Lrected upwards the - -NH2 and the - - H are directed towards the flavin group which lies at the side of the substrate. The FAD is probably bound to the enzyme through its phosphate and purine nucleotide groups (since the holoenzyme is fluorescent). Under the ravin, and masked by it, is an - - S H group of the enzyme 7. Some further light on the nature of the groups involved in the combination is provided by the effects of pH described in the following paper 16.

I t was mentioned earlier that there is some tendency for substrates with low reactivity to have high I / K m and vice versa. This may suggest that the rate-limiting step is the dissociation of the product from the holoenzyme (k+3 in Mechanism I), as has been already suggested by other authorsl~, 17, though without much experi- mental evidence. The rationale of this is that the substrate and product are not very different so far as binding groups are concerned, the affinity being largely determined by the R-group, so that substrates with a high affinity for the enzyme should give products which would tend to remain combined with the enzyme.

Biochim. Biophys. Acta, 96 (I965) 368-382


ACKNOWLEDGEMENTS

W e are g r a t e f u l to t h e R o y a l Soc ie ty for a g r a n t t o M. D. for t h e p u r c h a s e o f

t h e 0 3 e l ec t rode out f i t . One o f us (K. K.) is i n d e b t e d to t h e N o r w e g i a n Counci l for

Scient i f ic a n d I n d u s t r i a l R e s e a r c h for t h e a w a r d of a R e s e a r c h Fe l lowsh ip . W e wi sh

to t h a n k Mr. G. CATLIN a n d Mr. P. KENWORTHY for v a l u ab l e t e c h n i c a l ass i s tance .

W e are i n d e b t e d to Dr. P. K. TUBBS for g i f t s o f chemica l s .

REFERENCES

i E. C. WEBB, in M. DIXON AND E. C. WEBB, Enzymes, Longmans, London, 2nd Ed., 1964, p. 219.

2 H. A. KREBS, Biochem. J., 29 (1935) 162o. 3 A. E. BENDER AND H. A. KREBS, Biochem. J., 46 (195 o) 21o. 4 J. P- GREENSTEIN, S. M. BIRNBAUM AND M. C. OTLE'¢, J. Biol. Chem., 204 (1953) 3o7 • 5 M. WINITZ, M. BLOCK-FRANKENTHAL, M. IZUMIYA, S. M. BIRNBAUM, C. G. BAKER AND J. P.

GREENSTEIN, J. Am. Chem. Sot., 78 (1956) 2423 . 6 V. MASSEY, G. PALMER AND R. BENNETT, Biochim. Biophys. Acta, 48 (1961) I. 7 M. DIXON AND K. KLEPPE, Biochim. Biophys. Acta, 96 (1965) 357. 8 M. DIXON, Biochem. J., 55 (1953) 17o. 9 S. RATNER, V. NOClTO AND D. E. GREEN, J. Biol. Chem., 152 (1944) 119.

io A. H. NEIMS AND L. HELLERMAN, J. Biol. Chem., 237 (1962) PC 976. II G. R. BARTLETT, J. Am. Chem. Soc., 7 ° (1948) IOLO. 12 V. MASSEY AND Q. I'{. GIBSON, Federation Proc., 23 (1964) 18. 13 A. N. RADKAKRISHNAN AND A. MEISTER, J. Biol. Chem., 233 (1958) 444. 14 T. NAKAMURA, S. NAKAMURA AND Y, OGURA, J. Biochem. Tokyo, 54 (1963) 512. 15 M. BLANCHARD, D. E. GREEN, V. NOClTO AND S. RATNER, J. Biol. Chem., 163 (1946) 137. 16 M. Dixon AND K. KLEPPE, Biochim. Biophys. Acta, 96 (1965) 383 . 17 W. R. FRISELL, H. J. LowE AND L. I-tELLERMAN, J. Biol. Chem., 223 (1956) 75.


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d-amino acid oxidase II. Specificity, competitive inhibition and reaction sequence

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