Studies on uricase

514

BIOCHIMICA ET BIOPHYSICA ACTA

VOL. 2 2

(I956)

S T U D I E S ON U R I C A S E II. T H E ENZYME-SUBSTRATE COMPLEX* H A R O L D BAUM**,***, G E O R G H U B S C H E R * * , * * * AND H. R. M A H L E R

Institute [or Enzyme Research, University o[ Wisconsin, Madison, Wis. (U.S.A.) and Department o] Chemistry, Indiana University, Bloomington, Ind. (U.S.A.)

In the first paper of this series 1 we have reported on the isolation, purification, and characterization of uricase of high specific activity, and have presented evidence for the possible identity of the enzyme with a cuproprotein, containing one atom of metal per molecule of enzyme. It was also implicit in this presentation that the enzymebound copper ion was necessary for enzymic activity and provided a means of attachment of the substrate onto the enzyme. This supposition rested on the following evidence: (a) the copper content of the enzyme increases during the purification in a manner paralleling the increase in specific activity; (b) urate, like other purines is capable of chelating with metals2--among metal complexes and chelates those of copper are among the most tightly bound 2, 3,4; (c) the enzyme is inhibited by cyanide and by other metal-complexing agents which, like cyanide, are capable of acting as reducing agents as welll; (d) these inhibitions are either completely or partially overcome in the presence of urate 1, and (e) the spectroscopically distinct diethyl dithiocarbamate-copper-enzyme complex is dissociated in the presence of uratO. The present investigation is concerned with attempts at further elucidation of the structure of this enzymically active site and of the enzyme-substrate complex. The evidence to be reported is based largely on kinetic experiments involving the oxidation of uric acid by air in the presence of the enzyme and also of enzyme inhibitors such as urate (in excess), cyanide, purines related structurally to uric acid, and inorganic copper complexes. A spectrophotometric method was used for the determination of uric acid disappearance which is shown to be equivalent in certain cases to the measurement of the appearance of a short-lived intermediate (I) first discovered by PRAETORIUS 5. MATERIALS AND METHODS § Uric acid disappearance was measured spectrophotometrically in a m a n n e r described previously 1,s. The m e a s u r e m e n t of the short-lived intermediate (I) was also carried o u t s p e c t r o p h o t o m e t r i c a l l y * This investigation was s u p p o r t e d by a research grant, G-4128, f r o m the N a t i o n a l H e a r t I n s t i t u t e of the National I n s t i t u t e s of Health, Public Health Service, and a grant-in-aid of t h e American Cancer Society (on r e c o m m e n d a t i o n b y the C o m m i t t e e on G r o w t h of the National Research Council) to one of us (H.R.M.), as well as a grant-in-aid b y the N u t r i t i o n F o u n d a t i o n to Dr. DAVID E. GREEN. ** Post doctoral trainee of the National H e a r t Institute, United States Public H e a l t h Service. *** Present addresses, H.B.: 39 Sandon Road, B i r m i n g h a m , E n g l a n d ; G.H.: D e p a r t m e n t of Pharmacology, University of B i r m i n g h a m , B i r m i n g h a m , England. § The following abbreviations will be used: Az = optical density (log Io]I) at wavelength ~; tris = t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ; diol = 2,2-dimethyl-i,3-propanediol; e = molar ex-

Re]erences p. 527 .

VOL. 2 2 (1956)

STUDIES ON URICASE II

515

at 320 m#*, essentially as described by PRAETORIUS5. For all rate-runs a recording spectrophotometer, Beckman Model D U R was used; the runs were carried out at 25 °, and the data, where shown on figures, have been replotted using contracted axes. The initial rate, v, of the enzymic reaction is defined as the rate of disappearance of urate (or appearance of I) between 15 and 75 seconds. The preparation of uricase of high specific activity, and the definition of specific activity and other constants have been discussed in the first paper of this series 1. The substituted uric acids were highly purified samples from the Emil Fischer collection, made available to us through the courtesy of Prof. H. O, L. FISCHER. The pure purine derivatives were gifts of Dr. George B. BROWN. KINETICS OF ENZYME-CATALYZED REACTION Q u a l i t a t i v e observations

T h e r a t e of uric a c i d d i s a p p e a r a n c e , as m e a s u r e d b y t h e A A 29s, u n d e r s t a n d a r d a s s a y c o n d i t i o n s , in t h e p r e s e n c e of b o r a t e ions, is zero o r d e r w i t h r e s p e c t to s u b s t r a t e conc e n t r a t i o n u n t i l m o r e t h a n 6 0 % of t h e s u b s t r a t e i n i t i a l l y p r e s e n t has d i s a p p e a r e d . T h e n t h e r a t e a p p r o a c h e s a first o r d e r p a t t e r n . I t is also first o r d e r w i t h r e s p e c t to s u b s t r a t e at v e r y low uric a c i d c o n c e n t r a t i o n s ( < I.O- lO -5 M). I n t h e a b s e n c e of b o r a t e t h e r a t e a t a n y one p H a p p e a r s to be i n d e p e n d e n t of t h e n a t u r e of t h e buffer (see Fig. Ia). I t is also i n d e p e n d e n t of t h e ionic s t r e n g t h . I f r a t e m e a s u r e m e n t s are p e r f o r m e d in t r i s c h l o r i d e buffer, p H 8.0, a t ionic s t r e n g t h s of o . o i , 0.05, o.Io, 0.5 ° a n d I.O, i n i t i a l r a t e s s h o w i n g a s t a n d a r d d e v i a t i o n f r o m t h e m e a n of no m o r e t h a n t e n p e r c e n t a r e o b s e r v e d . S i m i l a r results are also o b t a i n e d in p h o s p h a t e or g l y c i n e buffers. T h e r a t e in b o r a t e , h o w e v e r , is a l w a y s m o r e r a p i d t h a n in a n y o t h e r buffer of t h e s a m e p H (see Fig. 2, E x p e r i m e n t s I a n d 5). If b o r a t e (final c o n c e n t r a t i o n ~ / O . o l M ) is a d d e d to t h e o t h e r buffers ( p h o s p h a t e , tris, b i c a r b o n a t e or glycine), e v e n at c o n c e n t r a t i o n s as h i g h as 0.5 M , t h e r e a c t i o n v e l o c i t y b e c o m e s i d e n t i c a l to t h a t o b s e r v e d in b o r a t e alone. IDENTITY OF REACTION IN PRESENCE AND ABSENCE OF BORATE I t a p p e a r e d desirable to d e t e r m i n e w h e t h e r t h e f o l l o w i n g t h r e e r e a c t i o n s p r o c e e d e d at t h e s a m e r a t e a n d t h u s c o u l d be e m p l o y e d i n t e r c h a n g e a b l y in k i n e t i c m e a s u r e m e n t s : (a) t h e d i s a p p e a r a n c e of u r a t e in t h e a b s e n c e of b o r a t e , (b) its d i s a p p e a r a n c e in t h e p r e s e n c e of b o r a t e a n d (c) t h e a p p e a r a n c e of I n t e r m e d i a t e I. T h r e e different app r o a c h e s to t h i s a i m w e r e e m p l o y e d . (a) T h e s p e c t r o p h o t o m e t r i c a l l y d e t e r m i n e d i n i t i a l r a t e of u r i c a c i d d i s a p p e a r a n c e , c o r r e c t e d for t h e a b s o r p t i o n of I n t e r m e d i a t e I at 293 mtz, c a n be s h o w n to be i d e n t i c a l w i t h t h e i n i t i a l r a t e of a p p e a r a n c e of I n t e r m e d i a t e I, a s s u m i n g e q u a l £max for b o t h uric a c i d a n d c o m p o u n d I*. (b) T h e v a r i a t i o n of t h e r a t e of uric a c i d d i s a p p e a r a n c e w i t h p H is s h o w n in Fig. I a for a v a r i e t y of buffers, all tinction coefficient; ES = enzyme-substrate complex; K M = Michaelis constant (observed); KES = pH-independent Michaelis constant; K I = enzyme-inhibitor dissociation constant; Kss = inhibitory enzyme-substrate complex dissociation constant; H2U = uric acid; H U - = urate-; U = ~ urate=; Vmax = rate at infinite substrate concentration (extrapolated); Vmax ~ maximal obtainable rate for any one variable (e.g. [U], [H+]). * For practical reasons it is desirable to measure the formation of Intermediate I at a wavelength at which uric acid does not absorb. 32o m/, has been chosen for this purpose 4. The true absorption maximum of this compound is at 305 m/~, but since the ratio e3os]e32o is known, determination of A 320 is tantamount to determination of A 30s. Thus the complete equality here under consideration is - ( A A 293 + 1.8 A A 220) = 2.32 A A 320, where the term on the left corresponds to the true rate of disappearance of uric acid, and the coefficients 1.8 and 2.32 are the ratios e~93/e320 and e305/e320 respectively for the pure Intermediate I (H. 1R. MAHLER, in preparation). Re/erences p. 527 .

516

H. BAUM, G. HUBSCHER, H. R. MAHLER

%

>.-

x

I-

~z

-~ I-

8o

uJ

O3

6O

< nD 2 03

._1 O3 n,-

(1956)

I00

o , .&j'

tJ <

x

VOL. 2 9

co..%_

\

\%

>

)40

O3 .J

20

o

I

<3 6 . 0

I

I

I

I0.0

8.0

I

/......t\

pH Fig. I a

6.0

\...

APPEARANCE OF I (UNCORRECTED) I I I I 8.0 I 0.0 pH

Fig. I b

Fig. ia. Dependence of rate of uric acid disappearance on n a t u r e and p H of buffer, o.o6/*moles of urate, 45 # m o l e s of the buffer at the p H indicated and 5 / , m o l e s borate adjusted to the a p p r o p r i a t e p H were used in 0.98 ml. At zero time 4 ~ of uricase of specific activity 5 ° was added to s t a r t the reaction. The optical density changes were m e a s u r e d continuously in the B e c k m a n D U R spectrop h o t o m e t e r against a b l a n k containing buffer and o . 0 4 / , m o l e s of urate per ml. The initial rate is t a k e n as the rate of u r a t e disappearance between io and 7° seconds; t e m p e r a t u r e - - 2 0 °. O = p h o s p h a t e ; • = tris; • = glycine. Fig. lb. I d e n t i t y of rate m e a s u r e m e n t s as m e a s u r e d b y u r a t e disappearance and b y the appearance of I. Reaction conditions were similar to those of Fig. I a b u t with borate o m i t t e d w h e n e v e r the appearance of I .500 -- ,I 2 was measured. Solid points ( 0 ) are d a t a for the disapFoTR, S. p, 8.2 pearance of u r a t e ; points (O) and dashed line those for the appearance of I m e a s u r e d at 320 m/,. W h e n corrected E for the first order rate of disappearance of I these d a t a .400 give rise to the solid line. oJ •~\

"-..... "-...

>-

~ .300 z

O3 a d


I.-

13_

o

•~ I

BORATE I

30

I

I

l

I

I

I

I

go 150 SECONDS

I

I

210

Fig. 2. I d e n t i t y of reaction p a t h in the presence and absence of borate. The reaction was m e a s u r e d continuously at 293 m # b y m e a n s of a recording s p e c t r o p h o t o m e t e r . I t was s t a r t e d at zero t i m e b y the addition of 3 7 of uricase of specific a c t i v i t y 6o. The lower solid line ( E x p e r i m e n t I) indicates the kinetics in tris (IOO /*moles) - borate (3.3 /*moles) buffer. F o u r s e p a r a t e e x p e r i m e n t s were performed in tris buffer (IOO/,moles) : In e x p e r i m e n t 2, o. 3 /*moles of KCN and 3.3/*moles of borate in 0.05 ml were added after 3 ° seconds, in e x p e r i m e n t 3, after 60 seconds and in e x p e r i m e n t 4, after 12o seconds. I n e x p e r i m e n t 5 (dotted line) no b o r a t e - K C N was added. All e x p e r i m e n t s in a total v o l u m e of i.o ml.

containing dilute borate. If these rates are recalculated on a relative basis and compared to the corresponding ones in the absence of borate but corrected for the rate of appearance of I, the data of Fig. Ib are obtained. It can be seen that if the rate of formation of I is corrected for its pH-dependent but non-enzymic rate of disappearance the two curves become superimposable. (c) The data of Fig. 2 demonstrate that the apparent slower rate of urate disappearance in the absence of borate is really due to the contribution of I to the A 293 and not an intrinsic rate difference. Cyanide in a concentration more than sufficient to stop the enzymic reaction completely (as determined in control experiments in the absence of borate when the A ~ga after this addition remains constant for periods up to five minutes) is added to the reaction at the Re#rences p. 527.

VOL. 2 2

(1956)

STUDIES ON URICASE II

517

different times indicated by the vertical arrows. Simultaneously borate is also added. The decay of the A 293 under these conditions must therefore be due to the disappearance of a component not identical with urate. This compound is I, whose decay at 320 m/~ under the same conditions can be shown to parallel exactly the disappearance of the non-urate material absorbing at 293 mtz. When this disappearance is complete (horizontal arrows ), the A 293, now corresponding to the true contribution of urate at the original time of addition of the cyanide-borate mixture, is found to be identical t o the A ~93 observed in a reaction mixture containing borate initially when allowed to react for the same length of time. Thus the true rate of urate disappearance (as distinct from the measured AA z93) is independent of the presence of borate. Kinetic data

The dependence of the kinetic constants of uricase on p H have been determined under Fig. 3- Variation of kinetic cons t a n t s with p H . The buffers and conditions indicated in the legend to Fig. i were employed. The enz y m e used was 2.0 ~, of specific activity 12o. The initial rate is defined as 2 × the rate of uric acid d i s a p p e a r a n c e between io a n d 4 ° seconds. F o u r different concentrations of urate were used, viz. o.oi, o.o2, 0.o 5 and o.08 # m o l e s in i.o ml. The readings at 293 m/t were taken c o n t i n u o u s l y against blanks containing 0.0o5, o.ooi, o.o 4 and o.07/~moles of u r a t e in the a p p r o priate buffer, t h u s keeping the A A 29a between e x p e r i m e n t a l and b l a n k to a value of o.ioo. The resuits were t h e n plotted for each p H b y the reciprocal m e t h o d of L I N E W E A V E R AND B U R K 11. These plots p e r m i t the evaluation of KM, the experimental, p H - d e p e n d e n t Michaelis c o n s t a n t which is t h e n 50 plotted as a function of p H (Fig. N 3a) and Vmax, the m a x i m u m veloci- O t y at infinite s u b s t r a t e c o n c e n t r a - _ X tion which plotted a g a i n s t p H is s h o w n in Fig. 3b. I n Fig. 3c the ~ 2.0 indicated function is plotted. I n x Fig. 3b the p o i n t s are the experi- m m e n t a l l y observed values, while the solid line has been calculated o from ~. 1.0

pH 8.0

9.0

I

I

I

I0.0

I

Ks

A

5.0

o x

T

o

2.0

o

o

.,,_I x ,V')

U

o

".-,'

o

L0

o

Vmax.

B

Vmox./K s

C

f-

2.0x

z,

1.0

~Tiila X

k3 E0 14,15,17 i + [H+]/KaEs + KbEs/LH+]

70

70

I

I

8.0

9.0

oH

I I0.0

I

I

I

I

7.0

8.0

9.0

IOD

pH

where kaE o is the m a x i m a l value obtained from the curve, and KaE s = IO-7"1 a n d KbES = lO -9"5. h a v e been calculated from it according to x4. Similarly in Fig. 3 c, the p o i n t s are experimentally determined, while the solid line is calculated from

k 3 Eo/ KES I + [H+]/KaE + KbE/[H+ ] w i t h the v a r i o u s c o n s t a n t s evaluated as before xa, i.e, KaE = io-7.~; KbF. = io -9.z. Vmax

KM

Re/erences p. 527 . 34

518

H. BAUM, G. HLIBSCHER, H. R. M A H L E R

VOL. 2 2

(1956)

a wide variety of experimental conditions and these results are summarized in Figs. 3a, b and c.

Inhibition by cyanide The strong inhibition of uricase by cyanide1, 6 has been re-investigated in some detail. It was found to be independent of enzyme concentration. If the logarithm of cyanide concentration is plotted vs. per cent inhibition at various pH's, the data of Fig. 4 are obtained. A family of sigmoid curves is observed and it will be seen that the extent of inhibition due to a particular cyanide concentration increases as the pH is lowered. If the concentration of cyanide necessary to effect a given degree of inhibition is plotted as a function of hydroxyl ion concentration a family of straight lines is obtained, whose slopes increase with increasing extent of inhibitory action (Fig. 5).

,'"''"

pH='ZO

°o 75%1~ib.~/

/ oH=

:~1 20

75

-w

i

=~ 50 I

-

V-

i

tll

Z

LO O

~ Q.

a5

/

/ ,,'

II

I,,

.

.

.

Ii

.

! J/ I

,,//

0.50 LOG (CNtota I

>Z

..

1.00 xlO 6)

I

I0

/ . /

.

Z 0 ~

,...oo 1.50

I

I

I I

I

50 I00 [OH" ] M xlO'

Fig. 5. Variation of cyanide concentration necessary for constant inhibition with pH. The data of Fig. 4 were transformed and re-plotted as indicated in the text.

Fig. 4- T h e e f f e c t of c y a n i d e c o n c e n t r a t i o n a n d p H o n e n z y m i c a c t i v i t y . S t a n d a r d a s s a y c o n d i t i o n s , s i m i l a r t o t h o s e of F i g . I w e r e e m p l o y e d , w i t h u r a t e a t 5.0. lO -5 M . S i x 7 of a n e n z y m e of specific a c t i v i t y 60 w e r e u s e d . P e r c e n t i n h i b i t i o n is d e f i n e d a s ( i - v c N / v ) × IOO w h e r e v a n d VCN a r e t h e i n i t i a l r a t e s i n t h e p r e s e n c e a n d a b s e n c e of c y a n i d e r e s p e c t i v e l y .

Inhibition by excess substrate The data of Fig. 6 show that the enzyme exhibits the phenomenon of substrate inhibition. The part of the bell-shaped curve reflecting this inhibition at high urate concentrations was found to be independent of enzyme concentration and, unlike the cyanide inhibition, insensitive to pH.

Inhibition by structural analogues o/urate Uricase has long been considered an enzyme of extreme substrate specificity. Thus KEILIN AND HARTREE6 had already shown that the methyl-substituted uric acids were incapable of acting as substrates but were competitive inhibitors, while VAN Re[erences p. 527 .

voL. 22 (I956)

STUDIES ON URICASE II

519

PILSUM~had d e m o n s t r a t e d t h a t x a n t h i n e was a good competitive inhibitor. I t appeared desirable to e x t e n d this list considerably, n o t only in the hope t h a t additional substrates m i g h t be found, b u t also to determine which analogues might act as effective competitive i n h i b itors. I n this m a n n e r considerable i n f o r m a t i o n concerning the active I00 site m i g h t be obtained. Fig. 7 is a LINEWEAVER-BURK8 plot of the 80 effect of a d d i t i o n of a n u m b e r of s u b s t i t u t e d purines on the rate of 60 urate oxidation b y uricase u n d e r s t a n d a r d conditions. I t shows all 40 the compounds tested to be competitive inhibitors. The i n f o r m a t i o n 20 o b t a i n a b l e from n u m e r o u s experi0 0 m e n t s of this sort is s u m m a r i z e d x 0 in Table I I which also includes >1>~ some d a t a on the specific oxidation I I I I I of urate b y copper ions 9 discussed 0.0 1.0 2.0 more fully in the n e x t paper. LOG (URATI: x 10s)

Fig. 6. The effect of uric acid concentration on rate. Standard assay conditions, using borate buffer pH 8.o and 6 7 of an enzyme of specific activity 6o, were used. The urate concentration was varied as indicated on the figure. The points are experimental points, the solid line is the calculated curve for 72

I

t'max

I + ~U~ + Ks--~

/i"M

KtlJ~

IO00

~

[U]

Y,

A

a 0 x io" t

e

~x~-'

C

~ xlo "6

D

t2XlO "$

E:

Z 3 X l O "s

F

4 0 X l O "5

G

l~XtO"4

Vmax is the maximal observed inital rate; K M

=

2.0"

IO - s M ;

/(ss =

4" I ° - 4 M .

O~ (M *t

Fig. 7. Competitive inhibition of uricase action by substituted purines. The reaction conditions Jl 0 were similar to those indicated for Fig. 3, except -I> that all runs were carried out in o.oi M borate 25O pH 8.0, and the urate concentrations shown on the figures were used. All inhibitors were used in a concentration of 4.0. IO-s M. The purines and the code employed are indicated in Table II. The Inhibitor-Enzyme Dissociation constants (KI) are calculated from v ' = I + KES(I EI]~ Vm~ [u] \ + K I ] "

I---- URATE I

75 =_ Ix

4

Inhibition by excess copper A d d i t i o n a l evidence concerning the active site comes from studies on the copper i n h i b i t i o n briefly alluded to in the first paper 1. I t is a p p a r e n t from Table I t h a t only in the presence of certain anions, viz. polyvalent ones capable of b i n d i n g more t h a n one copper atom, is this i n h i b i t i o n observed at all. The effect is e n h a n c e d b y the fact t h a t the c o m m o n cationic buffers usually employed in this p H range such as tris, diol or glycine, being s u b s t i t u t e d ammonias, all are capable of complexing with copper

Re]erences p. 527.

H. BAUM, G. HUBSCHER, H. R. MAHLER

520

TABLE INHIBITION

OF I N O R G A N I C A N I O N S

Buffer

0.02 M 0.02 M 0.02 M o.02 M o.o2 M o.o2 M o.o2 M o.o2 M 0.o2 M 2. i o -3 2. lO -3 2 . lO -8

5. 0 • l O - 4

5 . 0 . i o -4 I ,O' IO -4 1.0" 10 - 4

I .O" I O - 4

5.0. 5.0. 5.0. 5.0. 5.0" 5.0.

I

OF U R I C A S E B Y C O P P E R IONS IN T H E P R E S E N C E

C u + + (M)

IO-* IO-4 lO -4 lO-4 I o -4 lO - 4

5 . O. I O - 4

phosphate borate borate borate + borate + tris borate + borate + borate + M tris + M tris + M tris +

Per cent inhibition

i o -2 M t r i s 0.05 M tris 0.o 5 M 2. lO -3 o.oi M o.oi M o.oi M o.oi M

VOL. 2 2 ( 1 9 5 6 )

tris M tris tris carbonate carbonate carbonate

+ 0.2 M N a C l + 0.02 M borate

60 80 48 75 8 o io 63 12 80 75 ioo

All r u n s a t p H 8.0 a n d 25 °, i n t h e b u f f e r i n d i c a t e d , a t t h e c o p p e r c o n c e n t r a t i o n s h o w n . P e r c e n t i n h i b i t i o n is d e f i n e d a s ( i - vi/v) x i o o w h e r e v a n d vi a r e t h e i n i t i a l z e r o - o r d e r r a t e s i n t h e p r e s e n c e a n d i n t h e a b s e n c e of c o p p e r r e s p e c t i v e l y , i o ~ of a n e n z y m e of s p e c i f i c a c t i v i t y 5 ° w e r e u s e d a n d t h e - A A 293 w a s m e a s u r e d . TABLE INHIBITION

II

OF U R I C A S E BY U R I C ACID A N A L O G U E S

Compound added Substituents on purine ring

Code Position

A B C D E Urate F G H I J

2

-CI -CI -OH -OH -OH -OH -C1 -OH -NH 2 -NH 2 -NH 2

6

-C1 -C1 -NH -OH -OH -OH -NH -NH -NH -OH -NH

Inhibition per cent

8

2

2 2 2 2

I-methyl 3-methyl 7-methyl 3,7-dimethyl

-C1 -OH -NH -H -NH -OH -OH -OH -OH -NH -NH uric uric uric uric

Cu ++ catalyzed oxidation Rdative rate

2

94 90 90 7° 64

2 2

5o 46 20 o o

20o 60

44 37 22 o

20 15 0 o

2

acid acid acid acid

31 400 ioo 13 °

All e n z y m i c r u n s a t 25 ° i n t h e s t a n d a r d a s s a y s y s t e m , c o n t a i n i n g 0 . 0 2 M b o r a t e b u f f e r p H 8.0, w i t h b o t h u r a t e a n d i n h i b i t o r a t 4.0" I o - S M ; r e l a t i v e r a t e s , w i t h t h e r a t e f o r u r a t e e q u a l t o i o o . F o r e n z y m i c a s s a y s a n e n z y m e of s p e c i f i c a c t i v i t y 6 0 w a s u s e d ; f o r t h e C u ++ c a t a l y z e d r e a c t i o n 6 . 0 . lO -4 M C u S O 4 i n 0 . 0 2 M b o r a t e p H IO.O. All r a t e s a r e m e a s u r e d a s - A A 2 9 3 × r a i n -1.

quite strongly and thus making it unavailable for inhibitory action. Therefore the picture presented in Table I is a rather complex one involving competition between various complexing agents for the added Cu ÷÷. Although complexing of Cu ++ at other active sites m a y certainly be proposed as a n explanation for this inhibition, the added requirement for inorganic anions m a y be referable to the occurrence o5 reactions such as: Re]erences p. 527 .

voL. 22 (1956)

STUDIES ON URICASE II

521 O-

oE >Cu( OH

I /O = An--O

I

+ 2 A n = O + Cu ++ ~--- E > Cu ~HzO [ \O-

O-

An

k /OH )Cu(x O HzO

(I)

[ O-

i.e. the copper ion at the substrate-binding site, as well as the added copper is complexed, and thus made unavailable for enzymic action by the polyvalent anion. A more direct demonstration of this effect comes from the following experiment : An aliquot of enzyme in 1% carbonate was made 2.5" lO-4 M with respect to Cu ++ (A), two blanks were set up simultaneously--one containing another aliquot of enzyme in carbonate, but no copper (B) and one containing copper and carbonate but no enzyme (C). After standing at o ° overnight no precipitate had formed in any tube. The samples were therefore brought to pH 4.5 with glacial acetic acid and then adjusted to pH 7.2 by means of KOH. Tubes A and C now contained a clear solution while a strong opalescence was visible in tube B. After another 24 hours no precipitate was visible in A and C while blank B showed a precipitate of the enzyme. Occurrence of a reaction such as (I) would explain this phenomenon. The copper-enzyme-carbonate complex would have greater negative charge and therefore greater solubility compared to the ordinary enzyme at pH 7.2.

The e~ect o/oxygen tension The influence of oxygen on oxidation rates has already been studied by previous investigatorsS, 10. For this reason we have confined ourselves to a comparison of the kinetic constants with oxygen as the final acceptor, to those obtained in air under .50 : 4 5 0 0 c ol. standard conditions. The results of a typical experiment are : in air K m = 1.95. lO-5 M Vmax ----3.3o- IO-2 Fmoles × min -1; in oxyO .40 gen K M ---- 5.0" lO 4 M Vmax ----- 5.2"IO -~ Fmoles × min-1 (o.05 M glycine buffer pH ,% 9.0 containing 0.005 M borate ion. 3.0 7 .30 of an enzyme of specific activity 60 used as catalyst).

The effect o/temperature

.20

The effect of temperature on the rate of urate oxidation by uricase has been determined over a temperature range extending from 20 ° to about 60 ° . Measurements were performed in borate buffer at pH 8.0, by means of the recording spectrophotometer

9 .10

I = 1 2 , 4 0 0 cot. 3.001

3.00

.10

References p. 527.

i .20 1000IT

I

i .30

I

Fig. 8. T h e effect of t e m p e r a t u r e on reaction rate. S t a n d a r d a s s a y conditions, u s i n g b o r a t e buffer p H 8.0 a n d 2 ~ of a n e n z y m e of specific a c t i v i t y 12o. T h e initial rate is defined as z × .40 t h e r a t e of u r a t e d i s a p p e a r a n c e b e t w e e n i o a n d 4 ° seconds.

522

H. B A U M , G. H U B S C H E R , H. R. M A H L E R

VOL. 2 2

(1956)

with a cell compartment kept at the temperatures indicated. All reagents were brought to this temperature prior to addition of enzyme which started the reaction. A typical reciprocal plot of these data is given in Fig. 8. The activation energy for the enzymic reaction can be calculated b y the usual methods n to be 12,4oo cal. It is of interest to compare this value to an activation energy of approximately 14,ooo cal for the nonenzymic oxidation of uric acid catalyzed by copper ions 9. At more elevated temperatures the activation energy drops to 4500 cal which might indicate a different mechanism in this temperature range. If the temperature is increased further, denaturation of the enzyme takes place. DISCUSSION

As with any studies of mechanism and structure in enzyme catalyzed reactions based wholly or largely on kinetic evidence we can only arrive at permissible hypotheses and not at uniquely determined solutions 1°. Subject to these limitations a good deal of apparently consistent information can, however, still be derived from the observations here presented. The variation of the kinetic constants Vmax and K m with p H has recently been the subject of m a n y intensive studies12,13,14,15. LAIDLER especially has succeeded in systematizing the available information, in arriving at explicit steady-state solutions for a large variety of cases TM,in suggesting possible mechanistic interpretations, and in providing means for the calculations of the dissociation constants of the acidic and basic group (A and B) on the enzyme responsible for the typical bell shaped pH-activity plots observed in almost all cases, including the present one 17,1s. From the VmaJpH plot we can calculate the acid dissociation constants for the enzyme substrate

complex Ka(ES) and Kb(ES) in ALBERTY AND MASSEY'S notation, (i.e. ES "+I \ K~(Es) \ ES" \ Kb(Es) + H+

\

ES n-I) as 10 -7.2 and 10 -9'5. The corresponding constants for the free + H+

enzyme K~(eI and Kb(E) (E "+~ ~ a ( E ) ~ E" Kb(E) \ E,_~) can be obtained from the + + H+ H+ Vm~x/KM-pH plot and are found to be approximately I0 -7.5 and 10 -9.2. Of these two values that for the basic branch of the curve (Kb) m a y be in error b y as much as several tenths of a p H unit since no attempt was made to correct for effects due to ionization of the substrate (pK 1 = 5.4 which will not effect the results, but p K 2 = lO.219 which certainly may). With this proviso we might tentatively identify the two groups responsible for the two ionizations as an a-amino group (pK = 7-75) and an e-amino (or p h e n o l i c - O H ) group (pK = 9.5) respectively 3'4. From these observations and those discussed subsequently the following characteristic constants for the pure enzyme m a y be calculated (all in 0.02 M tris-borate buffer, temperature - - 2 0 ° , air) P/i'aE =

7.5

PKaEs

pKbE =

9.2

pKbEs = 9.5

=

7 .2

k3E o (pH-independent maximal velocity for 2.0 y of pure enzyme) = 3.3" lO-2/~moles × min -~ k3 (assuming molecular weight = 12o,ooo) = 167o × min-1; k3k(oxygen) 3 (air) - - 1.6 R e f e r e n c e s p . 527 .

v o L 22 (1956)

STUDIES ON URICASE II

523

KES ( p H - i n d e p e n d e n t Michaelis constant) = 1.7" lO -5 M K ~ (substrate " i n h i b i t o r " dissociation constant) = 4.o" IO -4 M AE (activation energy) = 12,4oo cal. The kinetic d a t a are consistent w i t h the enzyme b o u n d copper as the site for t h e a t t a c h m e n t of t h e substrate17,~3, 24. This site would p r o b a b l y have t h e s t r u c t u r e /OH /OH 2 _H+\ /OH E > Cu\OH~ for the t r a n s i t i o n E > CU\\oH2 ~ E > Cui\OH2 would p r o b a b l y occur /OH _H+\ in t h e p H range between 5 a n d 6 a n d the t r a n s i t i o n E > Cu\OH~ ~

E>

/OH CU\o H

in t h e range a b o v e p H lO.53, 2°. Some further s u p p o r t for this s t r u c t u r e comes from a consideration of t h e p H d e p e n d e n c e of t h e c y a n i d e inhibition (Fig. 4). I t is q u a l i t a t i v e l y obvious a n d can be d e m o n s t r a t e d q u a n t i t a t i v e l y using c o n v e n t i o n a l techniques of e n z y m e - k i n e t i c calculationsll, 31 t h a t if there be a release of h y d r o x y l ions d u r i n g the b i n d i n g of c y a n i d e b y t h e enzyme, a n d if c y a n i d e a n d u r a t e are b o u n d at t h e same site, the e x t e n t of c y a n i d e binding, a n d thus of t h e inhibition due to c y a n i d e will be inversely p r o p o r t i o n a l to h y d r o x y l ion c o n c e n t r a t i o n , as observed experimentally*. The i n h i b i t i o n b y excess s u b s t r a t e has also been t r e a t e d previously n, 33. R e g a r d less of the m e c h a n i s m assumed, w h e t h e r n o n - c o m p e t i t i v e or " u n c o m p e t i t i v e " t h e KES [urate7 r a t e law** can be shown to be of t h e form 22 Vma,/v = 1 + [urate] + Kss Vmax V

=

_

I

I + 2% / ~ . for K ~ > KBs this reduces to a p p r o x i m a t e l y KES Kp.s [U] ' i + ~ +

EU] K~

The d a t a of Fig. 6 are in a g r e e m e n t w i t h this i n t e r p r e t a t i o n . The K~, i.e. t h e dissociation c o n s t a n t for t h e complex c o n t a i n i n g enzyme a n d two molecules of u r a t e can be c a l c u l a t e d to be 4.0. IO -~ M. The fact t h a t this c o n s t a n t is p H - i n d e p e n d e n t m i g h t sug* The reaction with cyanide is pictured to proceed as follows

[E > Cu/OH]+ \/ O H J + CN- ~[

E>

/CN]++ Cu\oHj

OH-.

KCN

ka ha

[0H-]K' CN

Substituting into the rate-law for competitive inhibition, expressing KCN in terms of the observed constant K'CN, and the cyanide concentration [CN-] as a function of total cyanide added [CN] : and the dissociation constant for HCN, KHCN, we find that [CNJt = Y = a ([Urate] + KES) lO_14 KCN + KeN [OH-] ; a I where VCN and V are the inhibited and uninKHCN VCN hibited rates at any substrate concentration and pH. Under the conditions of the experiment, KES = 1.7" IO-~ and [urate] = 5.o. IO-~; KCN = 0.35. ** The expression for "'uncompetitive" inhibitionzl, i.e. binding of two molecules of substrate KM

at the same site is the one given. In the non-competitive case we have in addition a term ... + ~ .

Since experimentally/~M 2"5" IO-S and Kss = 4" IO-~ this last term is negligible. Thus no choice is possible between the two mechanisms UH E > Cu < U + H U - ~ E > Cu./ \ u uncompetitive K~s= k s and E > Cu < U + H U - ~ E > Cu < U non-competitive, k5 ~

UH Re]erences p. 527 .

524

VOL. 22 (1956)

H. BAUM, G. HOBSCHER, H. R. MAHLER

gest that the second molecule of substrate, like the first is bound to a site not undergoing proton ionization within the pH range tested, i.e. presumably the metal ion*. Additional information [® [ ® bearing on the structure of \ +/ the binding site on the en.Cu *"° zyme may be derived from O/° R2 , \ the nature and extent of the c competitive inhibition exerHN(0(siC <7,\ II + - - I ~l c a l GII ( ~ )<,,~:o--t~ + I II d-Rl"t(~ ted on enzyme action by HNH O-C. ~ / // 2,6,8-tri-substituted purines (Fig. 7 and Table II). If we write the chelate of urate [ ~ ~ i with the enzyme as indicated 1 11" I INHIBITOR-URICASE COMPLEX URA'i E-URICA,$E COMPLEX in Scheme I, i.e. between N- 7 SCHEME I and C-6"* (the structure suggested by ALBERT2 as the most reasonable one for these heterocyclic natural chelating agents), then it is reasonable to postulate the inhibitory enzyme-purine complexes to have similar structures. The difference in inhibitory efficiency of various purines

/

-

* This requiremen t also restricts the type of reaction permissible in the binding of substrate to the enzyme to one which does not lead to the net release of either hydrogen or hydroxyl ions. Since the urate mono-anion (HU-) is the species most abundantly present in the pH range under investigation the reaction would be /OH]+

E>Cu~:)H,J

ks. +H~O' Ks~-I'KEs=

k'\E>Cu< l kUa

+HU-

ks + ka kl

products recently determined that urate mono anion is ionized at N~. Thus the copper chelates of this compound will be stabilized by the following mesomeric forms : ** BERGMAN AND DIKST]~IN 19 h a v e I

I

/~XN/-

I

l~Ni

(-) ~,

(a)

I

(b)

I

t

(-~/ N( + ) ~

O-

I

~N__

~/N.

7~N//"

/~N

-<---->- ~_~ (+)>- o- <--->.

/ ~ N /

(f)

(c)

(e)

II

(+)~- O-

/ (--)

(d)

The corresponding structures for any purine with a substituent at C8 are then I

~/Nx /"/~| I N.x,~-R

I

<----~

-~/N\ (+) (-)T \ = R - * - - - - ~

I

R~/N\=(+) ]I ) (-)

[

(g)

I~.NI

t

(i)

(k)

(--)

(j)

I~NI (1)

Re]erences p. 527.

(h)

VOL. 22

(1956)

STUDIES ON URICASE II

525

having very similar or identical configurations around this site, must therefore be due to interaction of these compounds with the enzyme at additional binding sites and probably corresponds to interaction with the substituents at C-2 (site 3) and C-8 (site 2), on the purine ring. The following postulates, based on the structure of the enzyme-urate complex are sufficient to account for all the effects observed. In order to obtain effective binding to the enzyme a purine must possess: (a) a structure leading to high negative charge density at N-7; (b) no charge and a pair of unshared electrons at R,; (c) an electrophilic group at R 1 and (d) a strongly electrophilic group at R~ capable of forming strong hydrogen bonds. Compounds A and B (Table II) both will form complexes capable of strong binding at the two postulated sites on the enzyme; the electron affinity of chlorine .5 is greater than that of oxygen and thus A will have a greater tendency to fulfill conditions (a) and (c) above and will therefore be the better inhibitor. Structures such as (h) 8 are more likely with an amino-group at Cs than a hydroxyl group; therefore C and E are bound more tightly than is urate itself. F and G are very close structural analogues of nrate and are therefore bound as well as urate itself. The C = O at C8 has a (+)

(--)

greater tendency to exist as the resonance form C - - O than C = N H to exist as C - - NH. (+) (-) This tendency will lead to a decrease in stability of the complex of E when compared to C (as has already been mentioned structure (h) is more likely for the aminosubstituted Cs). We should expect the stability of the chelates to decrease in the order: no substituent at Cs and = 0 at Ce > - O H at Cs and = 0 at Ce > - O H at Cs and = N H , at Ce; this is the order observed for D (xanthine) > F > G . Compound H is of considerable interest: it is similar to G, an effective inhibitor, except for an amino group at C,. In the latter considerable contributions to the ground state will be made by structures such as I - I , ~ ,

and therefore it will be incapable of interaction according N (--) to postulate (d) with a group having considerable partial positive charge itself. H is therefore a poor inhibitor. Finally in I and J we have the last two effects superimposed i.e. less resonance in the Cs-imidazole system and a positive charge at C 2. Thus these compounds are not bound and therefore are not inhibitors. It is of interest to note that the nature of binding sites 2 and 3 here postulated purely on the basis of inhibitor studies is quite consistent with that proposed on the grounds of the p H dependence of the kinetic constants: a free amino group at 2 and a substituted ammonium ion or phenolic - O H at 3. In the non-enzymic Cu++-catalyzed oxidation of substituted purines condition (d) should not matter, less importance should attach to (c) while (a) and (b) become the over-riding considerations providing the compound in question is capable of donating electrons and being oxidized at all. The experimental results are in agreement with these predictions (Table II, last column). These findings suggested that 2,6-dihydroxy8-amino-purine might even be oxidizable in the presence of the enzyme. This is borne out by the data of Table I I I . This enzymic oxidation, slow in comparison to the rapid oxidation of urate, in conjunction with the other results presented permits us to draw certain conclusions with respect to the reactions subsequent to the formation of the enzyme-substrate complex, i.e. the actual oxido-reduction catalyzed by the enzyme. There are three Relerences p. 52 7.

526

H. BAUM, G. H/~IBSCHER, H. R. MAHLER

VOL. 2 2

(I956)

TABLE III I~VIDElgC~ FOR THE ENZYME-CATALYZED OXIDATION OF 2,6-DIHYDROxY-N-AMINO-PURINE Expt.

Conditions

5 ~ of 5 ~' of i o ~, of no

uricase, uricase, uricase, uricase,

reaction reaction reaction reaction

-zIA2sB

time time time time

-

60 io io io

min h h h

0.030 0.335 o.67o o.ooo

A M s o l u t i o n of p u r i n e in o.oi M t r i s buffer p H 9.0 in a i r a t 25 ° w a s us e d for all e x p e r i m e n t s .

mechanisms which might be operative to account for the great difference in the rate of oxidation of urate and the purine: I a n d I I S + Cu++ E III

S+Cu++E~---

kl " S Cu ++ E - -h z ~' S ' C u + E ~ 02--> P + C u

ISCu++E<----~S'Cu+EJ

+ + E + H~O 2

+02--> S C u E - - ks-+ P + C u + + E + H z O 2 I 02

S = substrate (urate or purine) ; S' = partially oxidized substrate, i.e. S - - I e - ; P = product = completely oxidized substrate, i.e. S - - 2e-. Mechanisms I and I I involve reduction of the enzyme-bound cupric to cuprous ion by urate, followed by its re-oxidation by oxygen, with the concomitant formation of product plus H~02, without the intervention of a ternary complex involving oxygen. The formation of the enzyme purine complex is rapid and reversible and cannot be rate determining, thus k3, k 4, k~ or k e must be rate-limiting. Mechanisms I and n differ in that in mechanism I k 3 > k4 while in mechanism I I k~ > kz. Mechanism I is unlikely since both urate and the purine are rapidly and catalytically oxidized by inorganic cupric complexes. Mechanism II, which is analoguous to that proposed b y CHANCE2e is unlikely because of the nature of the dependence of K M on O~ concentration. Both these mechanisms are also unlikely in the light of the composition of the enzyme and the stoichiometry of urate oxidation. The latter is a two-electron change, with no evidence of the formation of either a free radical intermediate, or a dimeric oxidation product occurring during the reaction. Thus there is no evidence that it might proceed in separated one-electron steps. The enzyme on the other hand only contains one atom of Cu per enzyme molecule and can only undergo one-electron changes. Thus mechanism I I I appears most likely and either k 5 or ke must be the rate-limiting constant. This mechanism is quite analoguos to that suggested by MICHAELIS~7 to account for the metal-catalyzed oxidation of cysteine and related compounds 1°. The mechanism here proposed implies that oxidation proceeds within a ternary complex involving copper-enzyme, oxidant and substrate. The metal is vital for attachment of the substrate by chelation, and as a means of transmission of electrons from it to oxygen. ACKNOWLEDGEMENTS

It is a pleasure to acknowledge the interest in and encouragement of this investigation by Dr. DAVID E. GREEN and m a n y helpful discussions with Dr. ROBERT A. ALBERT'/. Mr. GERmLLE COLMANOprovided capable technical assistance. R e / e r e n c e s p. 527 .

y o n . 22 (1956)

STUDIES ON URICASE II

527

SUMMARY O n t h e basis of kinetic a n d ancillary e x p e r i m e n t s a s t r u c t u r e for t h e e n z y m i c a l l y active site of ,H~O

uricase s u c h as E ~ C u ( \ OH

h a s been proposed. Similarly t h e s t r u c t u r e s of t h e e n z y m e - u r a t e ,

e n z y m e - c y a n i d e , a n d e n z y m e - e x c e s s s u b s t r a t e c o m p l e x e s m a y be f o r m u l a t e d as E ~ Cu ~ U,

> cu/C

/u

a n d E > Cu respectively. K i n e t i c c o n s t a n t s for t h e reaction w i t h u r a t e a n d \H20 UH a p p r o x i m a t e e q u i l i b r i u m c o n s t a n t s for t h e last t w o c o m p l e x e s h a v e been m e a s u r e d ; KES = 1.7" lO -5 M ; k a = 16oo × m i n -1 a t 2o °. T h e acid dissociation c o n s t a n t s of t h e groups on t h e e n z y m e c o m p r i s i n g t h e active site h a v e been d e t e r m i n e d f r o m t h e p H d e p e n d e n c e of t h e kinetic c o n s t a n t s KM a n d Vmax. A large n u m b e r of s u b s t i t u t e d p u r i n e s h a v e been s h o w n to be c o m p e t i t i v e inhibitors of t h e e n z y m e . This c o m p e t i t i o n is believed to t a k e place b y v i r t u e of chelation of t h e p u r i n e w i t h t h e e n z y m e in a m a n n e r a n a l o g o u s to t h a t proposed for urate. O n t h i s basis t h e 2,6-dihydroxy-8a m i n o p u r i n e - e n z y m e c o m p l e x s h o u l d m o s t resemble t h e e n z y m e - u r a t e c o m p l e x . This prediction h a s been verified since t h e p u r i n e c a n be oxidized b y o x y g e n in t h e presence of uricase, albeit e x t r e m e l y slowly. T h i s fact a n d kinetic d a t a s u g g e s t t h a t t h e r a t e - l i m i t i n g step d u r i n g uricase a c t i o n is t h e t r u e o x i d a t i v e step i n v o l v i n g oxygen, a n d t h a t a t e r n a r y c o m p l e x i n v o l v i n g e n z y m e , u r a t e a n d o x y g e n is p r o b a b l y i m p l i c a t e d . T h e a c t i v a t i o n e n e r g y for t h e reaction in t h e t e m p e r a t u r e r a n g e f r o m 25.0 to 38.0 ° is i2,4oo cal. REFERENCES 1 H. R. MAHLER, G. H/bBSCHER AND H. BAUM, J. Biol. Chem., 216 (I955) 625. A. ALBERT, Biochem. J., 54 (1953) 646. 3 I. M. KLOTZ, in W . D. McELRoV AND n . GLASS, The Mechanism o[ Enzyme Action, T h e J o h n s H o p k i n s Press, Baltimore, 1954, P. 257. 4 j . SCHUBERT, in R. N. GURD, Chemical Specificity in Biological Interactions, A c a d e m i c Press, N e w York, 1954, p- 114s E. PRAETORIUS, Biochim. Biophys. Acta, 2 (1948) 6o2. D. KEILIN AND E. F. HARTREE, Proc. Roy. Soc. (London), B 119 (1936) 114; ibid., 121 (1936) I73. 7 j . F. VAN PILSUM, J. Biol. Chem., 2o 4 (1953) 613. s H. LINEWEAVER AND D. BURK, J. Am. Chem. Soc., 56 (1934) 658. 9 N. GRIFFITHS, ]. Biol. Chem., 197 (1952) 399. 10 j . N. DAVlDSON, Biochem. ]., 32 (1938} 1386; ibid., 36 (1942) 252. 11 F. M. HOENNEKENS, in S. L. FRIESS AND A. WEISSBERGER, Techniques in Organic Chemistry, Vol. v i i , Interscience Publishers, N e w York, 1953, p. 53512 S. G. WALEY, Biochim. Biophys. Acta, IO (1953) 27. la M. DIXON, Biochem. J., 55 (1953) 161. 14 V. MASSEY AND R. A. ALBERTY, Biochim. Biophys. Acta, 13 (1954) 354x5 C. FRIEDEN AND R. A. ALBERTY, J. Biol. Chem., 212 (1955) 859. 16 K. J. LAIDLER, Trans. Faraday Soc., 51 (1955) 528. 17 K. J. LAIDLER, Trans. Faraday Soc., 51 (1955) 54 TM IS K. J. LAIDLER, Trans. Faraday Sot., 51 (1955) 55 TM lg F. BERGMANN AND S. DIKSTEIN, J. Am. Chem. Soc., 77 (1955) 691. 20 j . ]bJERRUM, Metal Ammine Formation in Aqueous Solution, P. H a a s e a n d Son, C o p e n h a g e n , 1941, P. 75. 31 H. L. SEGAL, J. F. KATCHMAR AND P. D. BOYER, Enzymologia, 15 (1952) 187. 22 B. H. J. HOFSTEE, J. Biol. Chem., 216 (1955) 235. 33 j . BOTTS AND M. MORALES, Trans. Faraday Soc., 49 (1953) 696. 24 M. MORALES, J. Am. Chem. Soc., 77 (I955) 4169. zs M. G. S. DEWAR, The Electronic Theory of Organic Chemistry, O x f o r d U n i v e r s i t y Press, 1949, p. 52. 26 B. CHANCE, in S. L. FRIESS AND A. WEISSBERGER, Techniques in Organic Chemistry, Vol. v i i , I n t e r s c i e n c e Publishers, N e w York, 1953, p. 665. ~7 L. MICHAELIS, in J. B. SUMNER AND K. MYRBACK, The Enzymes, Vol. II, P a r t z, A c a d e m i c Press, Inc., N e w Y o r k , 1951 . Received

March 23rd, 1956