The exchangeability of copper at active sites in ascorbic acid oxidase

ARCHIVES

OF

BIOCHEMISTRY

The

AND

BIOPHYSICS

Exchangeability Sites

From

338-347

99,

of Copper

in Ascorbic

RICHARD

,J. MAGEE

the Depnrimc’nt

of Chemistry,

(1962)

Acid

AND

Received

July

Oxidase

CHARLES

Columbia

at Active

University!

R. DAWSON New

Yock,

NW

I’ork

30,1962

The exchange reaction between the copper protein, ascorbic acid oxidase and radioactive CuB4 (cupric) ions has been reinvestigated under a variety of conditions. It has been confirmed that the exchange with the nonfunctioning (“resting”) enzyme is very much less t,han that observed with the functioning enzyme and is dependent on the specific activity. The very rapid ex&:mge observed with the functioning enzyme increases with the amount of substrate oxidized, and is a consequence of the catalytic mechanism rather than the processes by which the cnsyme becomes inact,ivated. It has been shown that the eschange occurs at copper sites involved in the enzyme activity and such sites remain active following the exchange. The results are discussrd in terms of different types of c’opper and copper bondings in the enzyme.

metal ions are present in the reaction medium (7). Recently, new evidence has been obtained indicating that the production of very small amounts of H202 by a secondary copper-catalyzed oxidation is responsible for the reaction inactivation (8). In the previous studies of exchange with radioactive copper ions, nonphysiological acidity produced by the rather high concent,ration of ascorbic acid may also have contributed to the severe loss of enzyme activity. As a consequence of this inact,ivat.ion, the earlier study did not permit a final conclusion as to whether the exchange of copper during the time when t.he enzyme was functioning was due t,o the catalytic mechanism of the enzyme or to the processes by which it became inactivated (3). The present investigat.ion was undertaken in an at.tempt to clarify this point. It has now been concluded that t,hc exchange is very fast and is in fact a consequence of the catalytic mechanism. The ascorbic acid oxidasc preparations employed in this study covered a wide range of specific activities, and the results obtained with them suggest two different types of copper (or copper hintl-

INTRODUCTIOK

The ascorbic acid oxidase from t.he yellow squash, C. pepo condensa, is known to be a copper prot,ein which in its purest form depends on firmly bound copper atoms for its activity (1, 2). When the enzyme functions, i.e., catalyzes the aerobic oxidation of L-ascorbic acid, the enzyme copper readily undergoes exchange wit,11 copper ions present in the reaction mixture (3, 4). Yrcvious investigators of this exchange reaction employed relatively large amounts of ascorbic acid. At the end of an exchange experiment the enzyme was essentially completely inactive. Such inactivation accompanying reaction (i.e., Lircact,ion inactivation”) has long been recognized as a characteristic feature of ascorbic acid oxidase behavior (5, 6). The reaction inactivation is known to be accentuated when copper, and to a lesser extent, other heavy‘Taken from a dissertation presented to the faculty of Columbia University by Richard J. Magee in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Present address: American Cyanamid Co., Princeton, N. 6. 338

-4SCORBIC

ACID

OXIDASE

ing) in the enzyme, the proportion varies with its purity.

AND

RADIOACTIVE

COPPER

of which

TABLE PROPERTIES

OF

THE

I ACID

ASCORBIC

PREPARATIOSS

Oxrn.4~~

EMPLOYEI)

EXPERIMENTAL Units

ENZYME

PREPARATION

ASD

ASSAY

The enzymes were prepared and characterized by procedures described elsewhere (9). The method of assay was essentially as described preI-iously (9), except that the positions of the enzyme and substrate during incubation in the Warburg flask were interchanged. As a result of this slight modification, the enzyme activities measured and reported here are 25% higher than would be found by the described method. The activity of homogeneous ascorbic acid oxidase has previously been found to be 750 units/pg. copper (2), as measured by the method of Powers et al. (6, 9). As measured by the modified method, homogeneous enzyme preparations should then possess about 950 units of act,ivity/pg. copper. The properties of the enzymes used are listed in Table I. The enzyme preparations were all extensively dialyzed against 0.1 111 acetate buffer prepared from triple-distilled water, recrystallized sodium acetate, and redistilled acetic acid. In one case (AA0 27Fa 10) the enzyme was passed through a cation-exchange column ( Amberlite lR-100 (Na’), see below) hcfore use in exchange studies. COPPER

ANALYSIS

A modification of the manometric micromethod developed by Warburg was used (10, 13). The method is very sensitive and, in fact, is limited to samples containing 0.05-0.3 pg. copper. This procedure was very satisfactory for analyzing the copper content of the purified enzymes (I) and in the standardization of copper solutions, such as the radiocopper solutions (*5%) (3). The method, however, was often erratic when applied to the reaction systems used in the copper exchange experiments. It is possible that the buffer salts or other reaction components in these samples interfere with the analysis, but attempts to modify the conditions of the manometric system so as to make the method more reliable were unsuccessful. The recovered copper values quoted herein are judged to be accurate to no bett,er than *lo%. The method of Gubler et al. (11) was investigated and employed for analysis of recovered copper in some of the rate studies. This too is a very sensitive method, but the desired precision was not obtained. THE

COPPER

In practically were carried out

EXCHAKGE

REACTIOS

all cases, the exchange reactions at room temperature in 4 ml. of

per pg. copper

of activity per mg. dry weight

Copper

% AA0 AA0 AA0 AA0 AA0 AA0 AA0 AA0 AA0 AA0 AA0 AA0

26 26 26 26 26 27 26 27 29 27 27 27

E Id E lc E 2 E lb C 3b Ii 2 C 3a C 7 55 Fa 10 K 1 F b

310 360 370 450 510 540 570 590 640 670 740 880

0.27 -

1370 1200 1200 2030 2180

0.27 0.25 0.31 0.26 0.23 0.18 0.18 0.27 0.23

acet,ate buffer (pH 5.7) in small flasks continually flushed with watersaturated oxygen. The 02 stream was flowed over the enzyme solution at a rate sufficient to cause gentle agitation of t,he surface. In a few cases the exchange reactions were carried out in st,andard 50-ml. Warburg flasks so that the course of the oxidation could be followed manometrically. In these cases, the flasks were flushed with 0,) sealed, and suspended in a constant temperature bath at 25.O”C. Except where otherwise noted, all experiments were carried out in 1.0 M acetate buffer. Variation of the buffer concentration from 0.3 to 1.0 M was found to produce no significant difference in the extent of exrhange provided that the pH remained close to 5.7. The reactants were added to the buffer in t,he following order: radiocopper, enzyme, and ascorbic acid (if used). Unless otherwise specified, the amount of radiocopper employed was equal to the copper content of the enzyme. Because very high concentrations of the enzyme were used, color changes could be observed during the reaction. The reaction mixture prior to the addition of ascorbic acid possessed the blue to green color characteristic of ascorbic acid oxidase. After adding the ascorbic acid, the solution berame colorless or yellow depending on whether the original enzyme was blue or green. The complete oxidation of the ascorbic acid was marked by the return of the original color to the system. The completeness of oxidation was also checked by titration with 2,6-dichlorobenzenoneindophenol (12) as well as by oxygen t)otals in those esprrimrnts in which manometers were used.

340

MAGEE REMOVAL

OF

AND

COPPER

IONIC

Following the exchange reaction, the enzyme was prepared for radioassay by quantitatively removing t,he non-enzymic ionic copper (including the extraneous radiocopper). This was accomplished by passing the enzyme solution through a column of a ration-xchange resin, Amberlite IR100 (Na’), as described previously (3). Radioassays and measurements of the residual enzyme activity were carried out as soon as possible after all the efAuents had been collected. In certain experiments the ionic copper was removed as its complex with ethylenediamine tetraon an anion-exchange resin, acetate (EDTA) Amberlite IR-4B (acetate form). The columns, 300 X 8 mm., were conditioned with four cycles of 5% HCl and 5% NH&OH. The last cycle was followed by 50 ml. of 5% acetic acid, 200 ml. of distilled water, and 50 ml. of 0.1 M acetate buffer, pH 5.7. RADIOCOPPER

AND

COUNTING

TECHNIQUES

The radiocopper, Cu” (tl,? = 12.8 hr.) was prepared by neutron bombardment of a copper wire in t,he atomic pile of the Brookhaven National Laboratory, Upton, L. I., New York. It was converted to Cu(N03)2 with nitric acid and diluted to the desired concentrations with acetate buffer. The copper concentration in the final dilution was checked by the Warburg method. Most radioactive samples were counted in a continuous flow, windowless Geiger-Mueller counter (Nuclear Instrument and Chemical Corporation) using an automatically timed scaling unit (Instrument Development Laboratories, Model 163). Samples were prepared as previously described (3) and were found to give a reproducibility of count between samples of *3%. All counting data were corrected for background count and for decay. Coincidence correction was found to be unnecessary in the range used. The percentage of the Cue” in the enzyme was calculated by comparing the counting rat,e of the enzyme sample to that of a “comparison standard,” TABLE PH

OF 4 ML. OF ACETATE ADDING ASCORBIC

II BUFFERS, ACID

PH

5.7,

(A&4)

PH After addmg AA

%z:

10 mg.

50 mg.

100 mg.

5.3 5.5 5.6 5.7

4.8 5.1 5.3 5.5

4.3 4.8 5.0 5.3

M

0.1 0.3 0.5 1.0

ON

DAWSON The comparison standard was a sample of t,he same mass, composition, and geomet,ry as the enzyme sample being counted, but which contained an amount of CP equivalent to the amount of Cu”’ initiallv . uqed (3). RESULTS EXCHAXGE ING

OF COPPER IN EXZYME-BUFFERED MIXTURES

THE FUNCTIOKREACTION

In order to eliminate changes in pH as a variable, 1 M acetate buffer was employed as the reaction medium for all the exchange studies reported below, unless otherwise specified. The effect of adding ascorbic acid to various concentraGons of acetate buffers is illustrated in Table II. The use of 1 M buffer assured that the enzyme would be maintained at its optimum pH throughout the reaction. No significant difference in exchange results was observed in three exchange experiments at buffer concentrations of 0.5, 0.75, and 1.0 M and which in all ot,her respects were identical. DEPENDENCE ON SUBSTRATE

THE AMOUKT OXIDIZED

OF

Several experiments were performed to determine how the copper exchange in a given amount of enzyme depended on the amount of substrate oxidized over a range of 2.5-25 mg. In each experiment the enzyme and ascorbic acid were allowed to remain in contact with radiocopper for the same time: that required for the complete oxidation of 25 mg. ascorbic acid. After the oxidation was complete, the reaction mixtures were passed through the cation-exchange columns, and bhe enzyme in the effluents was assayed for radiocopper. The dat,a presented in Fig. 1 show how the pcrccntage of radiocopper introduced into two different ascorbic acid oxidase preparations varied with the amounts of ascorbic acid oxidized. It is to be noted that as more ascorbic acid was oxidized, a higher l)roportion of radiocopper was introduced into each enzyme. The curves of Fig. 1 also show that

a considerable

alnount

of

radiocopper,

in respect, to the theoretical equilibrium value of 50%, was introduced into the enzymes when as little as 5 mg. ascorbic acid

ASCORBIC

/ 0

FIG.

I 5 ASCORBIC

on

thr

different act,ivities

I IO ACID

1. Showing

of radiocopper

the of

enzyme

of the

into

functioning

acid

oxidized

ascorbic Cu)

27 Fh),

I 20 (mg)

dependence

prrparations.

(units/pg.

OXIDASE

I 15 OXIDIZED

incorporated

amount

450 ; I?, (AA0

ACID

The are:

per

cent AS0

for

RADIO-1CTIVE

two

enzyme 26 E lb),

880. TABLE

RELATION

Ascorbic Sxpt acid oxi. No. dized

OF RADIOCOPPER

TO ENZYME

Components of exchange system hzyme prepn. No

ipecific &vit)

,T emp.

I

I

IE

“C. AA0 26 AA0 26 AA0 26 AAO27K2 AA0 27 AA0 27 7 8

III

INCORPORATION

-

AA0 AA0

E Id E lc’ E 2 Fa l( F b

26 E lk 27 K 2

PE.

311

COPPER

\vas oxidized. Under these circumstances little cnzynle inactivat,ion would be expeckd to occur. Early in this investigation, it was observed that the amount of radiocoppcr incorporated into functioning ascorbic acid oxidase varied from one preparation to another. The curves of Fig. 1 illustrate this point. There were some indicat,ions that the enzymes which incorporated the higher were those percentages of radiocopper which possessed t,he lower values of enzyme activity per microgram of copper, but the data do not, permit a definite conclusion to be drawn. It will bc noted, from Table III, that in these well-buffered systems with reasonably low ratios of ascorbic acid to enzyme, reaction inactivation was not significant,. In most cases, in fact, the amount, of enzyme activity recovered in the effluent was more than int’roduced to the exchange system. The percentage increase in act,ivity was greatest for those enzymes which initially had the lowest, activity per microgram of copper. One possible explanation is that such preparations may have contained significant. amounts of apoenzyme.

I 25

specific

A, (AA0

AKD

SPECIFIC Components ernuent”

ACTIVITY in

Cusr in enzyme (in eflluent)

’

fig.

UtzilS

units

a.

34 11.0 27 5.1 31 6.0 25 6.2 5.7 26 5.1 _______--

f dz 3~ + zt &

0.6 0.3 0.3 0.6 0.3 0.3

13.0 5.0 6.0 0.2 5.7 5.1

3,500 1,810 2,220 3,400 3,800 4,480

6,430 13 dz 1 30.4 2,8404.2 zk 0.430.6 4,820 31.2 3,350 ‘22.6 4,4005.1 + 0.530.2 3,3505.3 + 0.521.7

& f f 3.z + f

1.2 0.6 0.9 2.3 1.2’ 1.2

0.30 0.36 (0.31)d (0.22)” 0.33 0.21

25 25

xk 0.3 f 0.3

5.5 6.2

2,470 3,400

2,740 3,3501

* f

9.9 3.9

(0.44)d (0.30)d

5.5 6.2

L n Following removal of ionic copper by 6 0.75 M acetate buffer; all ot,hers in 1 c The theoretical equilibrium value for used is 500/,. d Copper determinations on the effluent that there was no change in enzyme copper. Warburg method (10, 13).

-I

-

:43.9 ~30.3

passage through an ion-exchange column. M acetate buffer. complete exchange when equivalent amounts were not made; these All copper values

values report’ed

are calculated in this table

of copper

are

on the assumption were made by t,he

342

MSGEE

AND

The results from tm;o experiments of this type, employing different specimens of ascorbic acid oxidase, are presented in Table IV. In the first stage CuG4 was incorporated into the functioning enzyme. In the second stage the labeled enzyme was separated int’o two portions. To each portion was added 2 equiv. of nonradioactive copper. but to only one portion was added ascorbic acid. Only in t.he experimenk where ascorbic acid was present, so that the labeled enzyme funct,ioned in the presence of nonradioactive copper, was there a decrease in the radioactivity of the enzyme as compared to the control sample. These results imply that the radiocopper initially taken up entered active sites of the enzyme and these sites remained active following the exchange. For such an interpretation to be strictly valid, it should be demonskated that the copper at,oms bound at sites which have become enzymically inactive are not, rcmovable when treated with ascorbic acid and copper ion. Attempts to prepare specimens of inactivated, Cu64-labeled ascorbic acid oxidase suitable for such a test were unsuccessful. Joselow, however, has rcported an experiment with inactivated enzyme which indicates that the copper in the reaction-inactivated enzyme is not easily

INCORPORATION OF RADIOCOPPER INTO ACTIVE ENZYME SITES DURING ENZYME FUNCTION

Additional evidence was sought as to whether the radiocopper incorporated into the functioning enzyme was situated at active or inactive sites. For this purpose the enzyme was subjected to two successive exchanges of copper. In the first, the enzyme was allowed to function as usual in the aerobic oxidation of ascorbic acid in the presence of radiocopper. As a result,, following passage through t,he cation-exchange column, the enzyme was labeled with radiocopper. The enzymes containing CuG4 introduced in this manner were then treated with additional ascorbic acid, this t,ime being allowed to function in the presence of nonradioactive copper. It was reasoned that if the CU”~ atom had initially entered during an inactivation step, being present now in an inactive site, it should no longer be excit,ed to exchange by ascorbic acid. If, on the other hand, the CP was present in an active site, it should be removable in the presence of ascorbic acid by exchange with nonradioactive copper. The latter would be a substantial argument in support of the view that exchange is associated wit,h changes in the enzymic site during the enzyme’s function. TABLE Cv6”

TWO-STAGE

EXCHANGE

T

SHOWING

THE

DAW’SON

IV INVOLVEMENT

OF ENZYMICALLY

First stage Further

AA0

26 E Id

(310 units/rg.

exchange of the Cu6’-labeled enzyme with nonradioactive CU

NOW

Enzyme CU

radioactive cu-+ ~__ wag.

Pg.

13.0

None

0.9

0.08 I

f

0.01

11.0

13.0

10

~ 4.0

‘0.36

f

O.Ol$

6.0

6.0

iTone

0.4

0.07

f

0.01

6.0

6.0

10

IO.30

f

O.Ol$

/a.

PK.

11.0

I

.S’TIWS

Second stage

Introduction of Cue* by exchange with the functioning enzyme

Enzyme preparation

ACTIVE

4scorbic acid oxidized

I --;y-

Final content Cl+

Final ratic+’

a.

;;

None 10

1.4 0.9

0.32 0.21

z!z 0.02 f 0.01

;:;

None 10

0.8 0.4

0.33 0.17

zt 0.01 f 0.01

Cu) I

AA0

26 E 2

(370 units/fig. -1

a Micrograms

I

of Cus4

I

1 1.8

I

I incorporated/pg.

of enzyme

copper

used.

ASCORBIC

ACID

OXIDASE

AND

removed (3). The enzyme was inactivated by continued reaction with ascorbic acid and then exhaustively dialyzed. Copper analysis of the final dialyzed enzyme showed that no copper had been lost on this treatment. Furthermore, on treating this inact,ivated enzyme with either ascorbic acid or dehydroascorbic acid and passing the mixture through a column of Amberlite IR-100, no loss of copper was observed. It appears justifiable, therefore, to assume that the copper of inactivated enzyme is bound so firmly as t,o be non-exchangeable. THE

RATE

OF

COPPER

EXCHANGE

TABLE EXCHANGE

E~XPERIMENTS

USING REMOVAL

A.40 AA0 AA0 AA0 AA0 AA0

Time of reaction before adding EDTA

Pt.

Pg.

mg.

rilin.

mg.

0 .2

8.4 8.4 8.4 5.1 5.1 4.3 4.3

0 0 0 10 10 10 10

0 150 155 0.25

4.1 4.1 4.1 5.0 5.0 5.0 5.0

27 C

n This

C Fb Fb Fa Fa

10 10

“resting”

AS

6.2 5.1 5.1 4.3 4.3 enzyme

was

in contact

with

EDTA

ANION-EXCHANGE

RESIS

FOR

Cu++

Ascorbic acid oxidized

Enzyme CU

27 27 27 27 27

AND

FREE

CU6”

EIlZyItX preparation

343

1’

EDTS OF

COPPER

copper not bound to the enzyme. A method meeting these requirements was devised employing ethylenediamine tetraacetate (EDTA) to combine with the non-enzyme cower, followed by the removal of the resultant copper-EDTA chelate on an anion-exchange resin. Passage through an anion-exchange resin also removes any unreacted ascorbic acid (16). All experiments were carried out in a 4-ml. reaction volume in 1.0 M acetate buffer, pH 5.7, under oxygen. An 80-200 molar excess of EDTA, based on the radiocopper used, was added at the appropriate time. At the end of the reaction time, the solution was allowed to percolat,e slowly through the anion-exchange column and was collected, with washings, to a volume of 25 ml. The ion-exchange resin was Amberlite IR-4B used in the acetate form. The results of several experiments testing this method and applying it to a preliminary rate measurement are presented in Table V. From these data several conclusions can be drawn. Ascorbic acid oxidase is not, inactivated by contact wit.h EDTA. Furthermore, it can be passed through an anion-exchange resin at pH 5.7 without loss of enzymic activity. CUDSEDTA was, however, removed quantitatively, either when in solution alone or in a mixture with resting ascorbic acid oxidase. The fourth and fifth experiments in Table V point up the effectiveness of EDTA in preventing free Cu64 in solut,ion from exchanging with the funct,ioning enzyme. In

Having thus obtained evidence that the exchange of copper during the functioning of ascorbic acid oxidase takes place at catalytically active sites which remain active following the exchange, we undertook to investigate, in a preliminary way, the process by which the exchange occurs. As is known from studies of metal chelates (14)) kinet,ic data can be used as criteria for distinguishing between unimolecular and bimolecular exchange mechanisms. All exchange reactions are first order (15)) but if the exchange is a bimolecular substitut,ion, the first-order rate constants are dependent on the concentrat’ions of the react,ants. In order to examine the rate of exchange of copper, a met,hod was required which would permit the exchange to be effectively halted at any desired time without injury to the enzyme and provide a means for the removal of the remaining

COPPER

RADIOACTIS’E

EDTA

for

180 min.

Total time of reaction

Per cent e”ZyIll.2 recovered

Per cent radiocopper incorporated into e*ZjTIlC

min. 104 -a 150 150 155 155

104 69 83 108 90

0 0 0.2 22.0 * 24.8 f 9.3 f

0.9 1.7 1.3

344

MAGEE

AND

the fourth experiment EDTA was added to the system containing ascorbic acid prior to adding the enzyme. After adding the enzyme, the mixture stood for 150 min., during which time all of the ascorbic acid (,lO mg.) was oxidized. The reaction mixture was then passed through an anionexchange column. Subsequent radioassay of the effluent showed only 0.2% of the total radiocopper to bc present for 60% recovery of t.he enzyme. For the similar fifth experiment, except that EDTA was not added until all the ascorbic acid was oxidized, 22% of t.he radiocopper was found for 83% recovery of the enzyme. A repeat of this experiment with a different enzyme resulted in 24.8% radiocopper incorporated for 108% recovery of the enzyme. These values are in satisfactory agreement with those obtained using the cation-exchange resin technique (cf. Table III, 10 mg. ascorbic acid). The rapidity of the copper exchange react,ion is indicated by the results of the seventh experiment in Table V. In this experiment the EDTA was added to the enzyme-Cu6” system 15 sec. after adding 10 mg. ascorbic acid. During the 15 sec., 9.3% incorporation of radiocopper occurred. In other words, nearly half of the anticipated radiocopper incorporation (based on the 155-min. period of the previous experiment,) act’ually occurred in the first 15 sec.

DAWSON

of the reaction. Such a high rate of exchange cannot be explained in terms involving an inactivated enzyme because the inact,ivation of the enzyme during its catalytic funct’ion is not that fast. In an attempt to get some information concerning the mechanism of the Cu’jl exchange with enzyme copper, the above technique of using EDTA to control the exchange reaction time was employed to determine the exchange half-lives at two different enzyme concentrations. The results are shown in Table VI. When the half-life values were plotted against the reciprocal of the enzyme copper concentration, the straight line through the two points intersectcd the origin. Such a result is best explained in terms of a bimolecular reaction involving a simple displacement, of an enzyme copper atom by collision with Cu”l ion (14). A mechanism involving an ionization and recombination of the enzymecopper chelate does not. fit the observed data because it would require that t,he halflife of the exchange be independent of the total enzyme concentration. THE

The “resting enzyme” refers, as previously mentioned, to the normal, nonfunctioning enzyme to which no ascorbic acid has been added. During the course of the

TABLE SHOWING

THAT

THE

tt/2

VALUES

OF THE

VI

OF EXCHANGE

EXCHANGIKG

EXCHAXGE OF COPPER IN RESTIXG ASCORBIC ACID OXIDASE

ARE

SPECIES

DEPEKDENT (ENZYME

ON THE

COSCENTRATIOKS

Cue”)

AND

Units of enzyme Enzyme

4.4 4.4 4.4

coppern

Cl9

Used

RWW ered

Irk?.

P&T.

2.0 2.0 2.0

2.0 2.0 2.0

1,280 1,280 1,280

675 G65 680

4.4 4.4 4.4

2,816 2,816 2,816

2255 2170 2370

f f f

0.1 0.1 0.1

Q Used AA0 29755; 0.5 M acetate buffer, 6 Determined by the method of Gubler c The maximum exchange under these d Obtained from the linear plot, of log

Cu recovered

4.6 4.8 4.5

f f f

PR.

sec.

-

15 30 60

7.5 11.5 12.2

33 52 56

54

15 30 60

10.5 14.7 17.7

48 67 80

25

O.l* 0.1* O.lb

10 mg. ascorbic acid. et al. (11). conditions using 10 mg. ascorbic (lOO‘% of maximum exchange)

sec.

acid was vs. time.

found

to be 22y0.

ASCORBIC

ACID

OXIDASE

AKD

present, work, it has been found, in agreement with previous investigations, that the exchange in the resting enzyme is very much slower than in the functioning enzyme. The dat,a observed for nine resting enzymes arc present,ed graphically in Fig. 2 and illustrate the dependence of exchange on specific activity. When resting enzymes were allowed to remain in contact wit,h radiocopper, the

;I/, , ,yxi,, 0

100 200 UNITS

300 400

500

OF ENZYME

600

700 600

PER pq

9001000

OF Cu

2.

24 TIME

COPPER

34.3

percentage of radiocopper incorporated was found to increase with time (Fig. 3). The enzyme which contained the higher specific activity (AA0 27 Fb, 880 units/rg. Cu) showed a linear relationship between t,he percentage of radiocopper incorporated and the time of contact. In contrast to this, the other enzyme preparation (AA0 27 FAlO, 670 units/pg. Cu) showed initially a higher rate of exchange, followed by a lower rate which became nearly parallel to t,hat of the purer preparation. These observations suggest, that t.wo types of copper may exist in the enzyme, i.e., act.ive, firmly bound! slowly exchangeable copper, and inactive, less firmly bound, more rapidly exchangeable copper. This suggestion is considered further in the .Discussion. DISCUSSIOhr

Showing the dependence of the extent of exchange of copper in resting AA0 on the specific activity per microgram of copper. Approximately 1 ecpuv. copper was used in every case. Times of contact are indicated as follows: (0) 1.5 hr.; (0) FIG.

RADIOACTIT’E

32

(HOURS)

FIG. 3. Showing the dependence of the exchange of copper in resting AA0 on the time of contact and the nature of the enzyme. AA0 27 Fa 10, 670 units/pg. Cu; AA0 27 Fb, 880 units/pg Cu.

Highly purified ascorbic acid oxidase in the resting state shows a negligible incorporation of radioactivity when equilibrat.ed with an equivalent amount of CuG4 for 1 hr. Only 6% of the CuG4 enters the enzyme when equilibrated for 28 hr. By comparison with the data in the literature for metal chelates, this suggests (17, 18) that in the resting state the copper of ascorbic acid oxidase is held in a polydentate ligand. The copper exchange process in functioning ascorbic acid oxidase has been found to be associated with the changes which the enzyme undergoes in performing its catalytic function and not, to be dependent on the reaction inactivation process. Evidence has been obtained t.hat, this exchange is very rapid, and that the sites at which it occurs continue to be enzymically active after the exchange has taken place. The exchange appears t,o occur by a bimolecular process involving copper, such as the displacement. of one copper atom by another via a collision mechanism. Despite the fact that the exchange is rapid, equilibrium of exchange is established relatively slowly. This is evidenced by the fact that the total amount of radiocopper incorporated into the enzyme increases as the amount of ascorbic acid oxidized increases. This suggests that certain of the active sites are more accessible

346

MAGEE

AKD

than others; only at the higher concentrations of substrate do essentially all of the copper atoms become involved in exchange. Since it is likely that there is a reversible change in the oxidation state of the enzyme copper during its catalytic function (Cu(II)AAO F?: Cu(I)AAO), the rapid exchange reaction is undoubtedly a direct result of such a transformation. A possible explanat,ion of t,his transformation has been offered previously (3). It was suggest.ed that t#he copper in the resting enzyme is in the Cu(I1) state and exists in a structure (possibly square, coplanar) in which the copper to protein bonds are relatively strong. The reduced enzyme produced during t,he oxidation of the subst,rate contains Cu(1) less firmly held to the protein by virtue of weaker copper to protein bonds in a tetrahedral configuration. It is interesting to note that in complex ions, the exchange of the metal atom is invariably much faster for the lower state of oxidation of the metal than for the higher state of oxidation (19-21). Because of the reducing action of ascorbic acid, present in relatively high concentration, the labeled copper ions in the functioning enzyme-substrate system are very likely in the Cu(1) form. The exchange, then, probably involves the displacement of G(I) atoms from the protein by Cu(1) ions from the solution. After the Cu(1) has been bound to the protein, it is reoxidized to the Cu(I1) state by molecular oxygen and the enzymic site is ready to function again. To explain the observed differences of copper exchange rates for ascorbic acid oxidase preparations of various specific activities, it must be supposed t,hat the copper atoms in the enzyme vary considerably in their mode or extent of binding. Some are very easily displaceable from the resting enzyme, while others are displaceable only while the enzyme is undergoing reaction. A low value for the activity per microgram of copper of an enzyme indicates that the enzyme contains copper in excess of t,hat at the active sites. It might then be supposed that it is t.his inactive copper which, although nondialyzable, is readily

DAWSON

displaced by CuG4 ion in the resting state. Several theories may be suggested for the difference in t,he exchangeability of active and inactive copper atoms. It might be that the inact,ive copper is stabilized in a state of oxidation different from that in the active enzyme. It might also be t,hat t,here is a lower degree of chelat,ion of the copper by the protein at the inactive sites, or t,hat the stereochemistry of the inact,i\-e copper is more favorable to exchange. In connection with the proposal of two types of copper in ascorbic acid oxidase, it is pertinent to note some observations on another copper enzyme. The exchange of copper in resting and functioning tyrosinase (polyphenol oxidase) has been found to depend on the specific activity of t,he tyrosinase preparations in a manner suggesting that there are two types of copper (22). In polyphenol oxidase, it has been found previously by Kubowitz that the copper, even though entirely nondialyzable, could occupy inactive as well as active positions in the enzyme (23). An attractive hypothesis is that the enzymically inactive copper in those enzymes which possess low activity per microgram of copper is present in the CuiI) form even in the resting state. It may be st,abilized in this form by virtue of some extraordinary type of chelation by the protein. By means of Q, a’-dipyridyl we have been able to demonstrate qualitatively the presence of a significant amount of Cu (I) in resting ascorbic acid oxidase of low specific activit,y. This reduced copper then undergoes exchange with the added radioactive copper ions at a faster rate than the copper present in the pot,ent.ially active sites of the resting enzyme. The exchange rate is still slower than in the funct,ioning case, possibly because the copper ions in solution are Cu (II) rather than Cu (I). Upon t.reatment with ascorbic acid under the conditions employed in the exchange experiment, the enzyme appears t,o undergo changes, one result of which is the rcst,oration of function to previously inactive sites. Such activation was actually observed (Table III, Exps. l-3). This activation may involve st.ructural changes in t,he pro-

ASCORBIC

BCID

OXIDASE

tein (e.g., reduction of disulfide bonds). As a result the formerly inactive copper is once again able to undergo the reversible oxidation-reduction essential t,o the enzymic activity. Further investigations of the nature of the copper in ascorbic acid oxidase are in progress in these laboratories. ACKNOWLEDGMENTS Portions of this work were carried out under grants from The Eli Lilly Co. and the Nutrition Foundation. One of us (R.J.M.) is especially indebted t.o the Lalor Foundation for a grant which permitted a summer’s research at, the Brookhaven Laborat,ories arranged and carried out with the aid of Drs. R. Anderson and R. M. Dodson. The assistance of Stanley Lewis in the purification of thr enzyme is acknowledged with sincere appreciation.

AND

RADIOACTIVE

R., Arch.

9.

10. 11.

12.

13. 14. 15. 16. 17.

REFERENCES 1. LOVETT-JAKISOX, P. L., .~ND NELSOS, J. M., J. Am. Chem. Sot. 62,1409 (1940). 2. DUNN, F. T., AXD DAWSON, C. R., J. Biol. Chcm. 189,485 (1951). 3. JOSELOW, M., AND DA~SOX, C. R., J. Biol. Chem. 19&l, 11 (1951). 4. DAWSON, C. R., i?t “Copper Metabolism,” p. 18. The Johns Hopkins Press, Baltimore, 1950. 5. STEINMAN, H. G., AND DAWSON, C. R., J. Am. Chem. Sot. 69, 1212 (1942). 6. POU’ERS, W. H., AXII DAWSON, C. R., J. Gen. Physiol. 27, 167,182 (1943).

18.

19. 20.

21.

22. 23.

315

N., MAGEE, R. J., AND DAWSON, C. Biochem. Biophys. 81,135 (1959). TOKUYAMA, K., AND DAWSON, C. R., Biochim. et Biophys. Acta 56,427 (1962). Dawso~, C. R., AND MAGEE, R. J., in “Methods of Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.). Vol. II, p. 381. Academic Press, New York, 1955. WARRGRG, O., Rio&em. Z. 187, 255 (1927). GUBLER, C. J., LAHEY, M. E., ASHENBRUCKER, H., CARTWRIGHT, G. E., ;ZND WINTROBE, M. M., J. Biol. Chem. 196,209 (1952). BESSEY, 0. M., J. Biol. Chem. 126,771 (1939). WARBGRG, O., .~XD KREBS, H. A., Biochem. Z. 190,193 (1927). DGFFIELD, R. B., .~ND CALVIX, M., J. Am. Chcm. Soc.68,557 (1948). MCKAY, H., Nature 142,997 (1938). JACKEL, S. S., MOSBACH, E. H., AND KING, C. G., Arch. Biochem. Biophys. 31,442 (1951). RUBEN, S., KAMEN, M. D., ALLEN, M. B., .UD NAHIXSKY, P., J. Am. Chem. Sot. 64, 2297 (1942). MARTELL, A., .4x11 CALVIS, M., “Chemistry of Metal Chelate Compounds,” p. 207. PrenticeHall Co., New York, 1952. JONES, S. S., AXD LONG, F. A., J. Phys. Chem. 56,25 (1952). RICH, R. L., ASD TAUBE, H., J. Phys. Chem. 58,6 (1954). WEST, B., J. Chem. Sot. 1954, 395. DRESBLER, H., .~XD DAWSOS, C. R., Biochim. et Biophys. Acta 45,508,515 (1960). KUBO~ITZ, F., Biochem. Z. 299,32 (1939).

7. BENHAMOU, 8.

COPPER