Oxidation of Organic Compounds - ACS Publications

The enzyme from bovine adrenal glands contains. Cu2+ and a smaller amount of Cu+. When the enzyme oxidizes ascorbate to dehydroascorbate, most of the ...
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73 The Mechanism of Action of Dopamine β-Hydroxylase S E Y M O U R K A U F M A N , W I L L I A M F . BRIDGERS, and JOSEPH B A R O N

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1

2

Laboratory of General and Comparative Biochemistry, National Institute of Mental Health, U . S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Bethesda, Md. 20014

Dopamine β-hydroxylase catalyzes the side-chain hydroxylation of dopamine and other phenylethylamine deriva­ tives. Ascorbic acid serves as a specific electron-donating cofactor. The enzyme from bovine adrenal glands contains Cu and a smaller amount of Cu . When the enzyme oxidizes ascorbate to dehydroascorbate, most of the Cu is reduced to Cu . Added substrate is hydroxylated, and Cu is reoxidized to Cu . This indicates that most of the protein-bound Cu undergoes cyclic reduction and oxidation during hydroxylation. The results also rule out an oxygen­ -carrier function for ascorbate. The possibility that α β­ -substituted hydroperoxide of the substrate is formed as an intermediate in the reaction has been examined with the use of β,β'-tritium-labelled substrate. The results indicate that such an intermediate is unlikely. 2+

+

2+

+

+

2+

2+

"p\opamine ^-hydroxylase catalyzes the hydroxylation of dopamine ( 3,4-dihydroxyphenylethylamine ) to norepinephrine according to Reaction 1 (14). Dopamine + 0

2

+ ascorbate —» norepinephrine + H 0 + 2

dehydroascorbate (1)

W i t h catalytic amounts of enzyme, the hydroxylation reaction is specific for derivatives of ascorbate (14). The enzyme is relatively non­ specific for the substrate; the minimum structural requirements appear to be a benzene ring with a two-carbon side chain that terminates i n an amino group (2, 3, 6, 15). The enzyme activity is stimulated markedly Present address: Department of Medicine, University of Miami School of Medicine, Miami, Fla. 33136. Present address: University of Chicago Medical School, Chicago, Ill.

1

2

172 In Oxidation of Organic Compounds; Mayo, F.; Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

73.

Dopamine

KAUFMAN ET AL.

173

^-Hydroxylase

b y c e r t a i n d i c a r b o x y l i c acids s u c h as f u m a r a t e a n d a-ketoglutarate

(14).

R e c e n t l y , other h y d r o x y l a s e s h a v e b e e n r e p o r t e d to s h o w a s i m i l a r d u a l r e q u i r e m e n t f o r ascorbate a n d a d i c a r b o x y l i c a c i d (1, 7, 8,

13).

D o p a m i n e /^-hydroxylase has b e e n o b t a i n e d i n essentially p u r e f o r m f r o m b o v i n e a d r e n a l glands (4).

It has a m o l e c u l a r w e i g h t of a p p r o x i ­

m a t e l y 290,000. A l t h o u g h the p r o t e i n is colorless, m e t a l analysis s h o w e d that it is a c o p p e r p r o t e i n c o n t a i n i n g b e t w e e n 0.65

a n d 1.0 jugram of

c o p p e r p e r m g . of p r o t e i n ( 4 - 7 moles of c o p p e r / m o l e p r o t e i n ) . P a r t of the c o p p e r is present as C u a n d p a r t as C u .

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+

Cu

+

Cu

2 +

2 +

A l t h o u g h the a m o u n t of

varies f r o m one e n z y m e p r e p a r a t i o n to another,

the

amount

of

is r e l a t i v e l y constant a n d is e q u a l to a b o u t t w o moles p e r m o l e of

enzyme. T h e c o p p e r is essential f o r e n z y m e a c t i v i t y . It c a n b e r e m o v e d b y t r e a t i n g the e n z y m e w i t h c y a n i d e a n d a m m o n i u m sulfate, r e s u l t i n g i n a n a p o e n z y m e w i t h o u t detectable h y d r o x y l a s e a c t i v i t y . C o p p e r is the o n l y c a t i o n tested w h i c h c a n restore a c t i v i t y to the a p o e n z y m e . In

the absence of substrate,

the e n z y m e c a n r a p i d l y o x i d i z e a n

e q u i v a l e n t a m o u n t of ascorbate to d e h y d r o a s c o r b a t e .

T h e other p r o d u c t

of this r e a c t i o n is a r e d u c e d f o r m of the e n z y m e i n w h i c h most of the Cu

2 +

has b e e n r e d u c e d to C u . T h e r e d u c t i o n of the e n z y m e b y ascorbate +

c a n take p l a c e a n a e r o b i c a l l y .

W h e n the r e d u c e d e n z y m e is exposed to

substrate a n d o x y g e n i n the absence of ascorbate,

p a r t of the C u

+

is

r e o x i d i z e d to C u , a n d a n e q u i v a l e n t a m o u n t of substrate is c o n v e r t e d 2 +

to h y d r o x y l a t e d p r o d u c t .

T h e valence

changes

i n the p r o t e i n - b o u n d

c o p p e r h a v e b e e n o b s e r v e d w i t h b o t h a c o l o r o m e t r i c m e t h o d (4) w i t h E S R measurements

and

(5).

B a s e d o n these results, the h y d r o x y l a t i o n r e a c t i o n has b e e n f o r m u ­ l a t e d (4,

as s h o w n i n R e a c t i o n s 2, 3, a n d 4, w h e r e R H stands f o r

5)

substrate a n d R O H for h y d r o x y l a t e d p r o d u c t . E-(Cu ) 2 +

E-(Cu ) +

2

2

+ ascorbate —» E - ( C u ) + dehydroascorbate + 2 H +

+ 0

E-(Cu ) -0 +

2

2

2

E - ( C u ) + R O H + H Q +

(2)

2 +

2

(4)

2

T h e scheme is u n d o u b t e d l y o v e r s i m p l i f i e d . It is possible, for ex­ a m p l e , that r e d u c t i o n of the e n z y m e b y ascorbate i n v o l v e s the f o r m a t i o n of s e m i d e h y d r o a s c o r b a t e a l t h o u g h d e h y d r o a s c o r b a t e is the o n l y p r o d u c t that has so f a r b e e n d e t e c t e d .

It is also possible that R e a c t i o n 3, the

c o m b i n a t i o n of o x y g e n w i t h the e n z y m e - b o u n d c o p p e r , does not o c c u r i n t h e absence of substrate.

It is of interest i n this r e g a r d that the r e d u c ­

t i o n of the e n z y m e b y ascorbate occurs r a p i d l y i n the absence of substrate. T h i s is i n contrast to results o b t a i n e d w i t h salicylate h y d r o x y l a s e , w h e r e r a p i d r e d u c t i o n of the presence of substrate

enzyme-bound

flavin

by D P N H

requires

(9).

In Oxidation of Organic Compounds; Mayo, F.; Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

the

174

OXIDATION OF ORGANIC COMPOUNDS

III

Reactions 2, 3, a n d 4 t e l l us little a b o u t h o w o x y g e n is a c t i v a t e d d u r i n g the h y d r o x y l a t i o n r e a c t i o n , a n d at the m o m e n t one c a n o n l y speculate a b o u t the details. T h e scheme a n d the results o n w h i c h it is based

d o , h o w e v e r , r u l e out

several general

types

of h y d r o x y l a t i o n

m e c h a n i s m . T h e fact that the e n z y m e c a n b e r e d u c e d a n a e r o b i c a l l y b y ascorbate to a f o r m w h i c h a c t i v e l y supports substrate h y d r o x y l a t i o n i n the absence of ascorbate rules out a n y m e c h a n i s m for this

enzyme-

c a t a l y z e d r e a c t i o n i n w h i c h the ascorbate f u n c t i o n s as a n o x y g e n carrier. S u c h a role has b e e n p o s t u l a t e d for t e t r a h y d r o p t e r i d i n e s (16),

which

c a n serve as specific e l e c t r o n - d o n a t i n g cofactors (just as ascorbate does

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w i t h d o p a m i n e /^-hydroxylase) i n c e r t a i n a r o m a t i c h y d r o x y l a t i o n reac­ tions

(10,12).

W e h a v e i n the past c o n s i d e r e d another t y p e of h y d r o x y l a t i o n m e c h a ­ n i s m ( R e a c t i o n s 5 a n d 6) that i n v o l v e s the f o r m a t i o n of a h y d r o p e r o x i d e of the substrate as a n i n t e r m e d i a t e , f o l l o w e d b y r e d u c t i o n of the p e r o x i d e to the h y d r o x y l a t e d p r o d u c t (11).

A similar mechanism involving

the

f o r m a t i o n of a t r a n s a n n u l a r p e r o x i d e of the substrate has also b e e n p r o ­ posed recently for aromatic hydroxylations RH + 0

2

(18).

-> R O O H

(5)

R O O H + ascorbate - » R O H + dehydroascorbate + H 0

(6)

2

I n the l i g h t of the results d i s c u s s e d earlier o n the r e d u c t i o n of the e n z y m e b y ascorbate, this o l d scheme w o u l d h a v e to be m o d e r n i z e d b y r e p l a c i n g the ascorbate i n R e a c t i o n 6 w i t h the r e d u c e d e n z y m e , E - ( C u ) . +

2

In

this m e c h a n i s m , it is a s s u m e d that the o n l y role of the e n z y m e - b o u n d c o p p e r is to a l l o w transfer of electrons f r o m ascorbate to the h y d r o ­ p e r o x i d e of the substrate. W e h a v e t r i e d to detect the f o r m a t i o n of a h y d r o p e r o x i d e of the substrate i n the absence of t h e o v e r - a l l r e a c t i o n b y u s i n g a

substrate

l a b e l l e d w i t h t r i t i u m i n the /? positions. (7)

If R e a c t i o n 7 o c c u r r e d , t r i t i u m s h o u l d b e released i n t o the m e d i u m i n the absence of ascorbate.

Sufficient e n z y m e w a s u s e d i n these experiments

to detect R e a c t i o n 7 e v e n if the h y d r o p e r o x i d e f o r m e d d i d not

exceed

the a m o u n t of e n z y m e present. B e f o r e experiments i n the absence of ascorbate w e r e p e r f o r m e d , the extent of d i s c r i m i n a t i o n against the t r i t i u m - l a b e l l e d substrate d u r i n g the h y d r o x y l a t i o n r e a c t i o n w a s estimated. T h e h y d r o x y l a t i o n of / ^ ' - t r i t i u m l a b e l l e d t y r a m i n e was s t u d i e d as a f u n c t i o n of t i m e .

T h e reaction was

f o l l o w e d b y m e a s u r i n g the a m o u n t of o c t o p a m i n e ( 1 - [ p - h y d r o x y p h e n y l ] -

In Oxidation of Organic Compounds; Mayo, F.; Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

73.

K A U F M A N

E T

Dopamine

A L .

175

^-Hydroxylase

2 - a m i n o e t h a n o l ) f o r m e d a n d the a m o u n t of t r i t i a t e d w a t e r released.

The

results ( T a b l e I ) i n d i c a t e that the isotope effect is a b o u t 1.8. T h e results of e x p e r i m e n t s i n T a b l e II.

About 0.2%

d e s i g n e d to d e t e c t R e a c t i o n 7 are s h o w n

of the t r i t i u m i n i t i a l l y present i n the substrate

w a s v o l a t i l e u n d e r c o n d i t i o n s of l y o p h i l i z a t i o n . I n the a b s e n c e of ascor­ bate, n o e n z y m e - d e p e n d e n t

release of t r i t i u m w a s

detectable.

In

the

p r e s e n c e of ascorbate—i.e., w h e n the o v e r - a l l h y d r o x y l a t i o n r a c t i o n t o o k

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T a b l e I. Incubation Time, min.

Time-Course

of the Conversion of Tyramine Tritium Release, CPM/0.05 ml.

Octopamine Formed, pmoles

5 10 20 40

to Octopamine"

0.111 0.219 0.414 0.685

Found

Expected

Isotope Effect

258 498 913 1754

470 928 1760 2900

1.82 1.86 1.92 1.65

A l l tubes contained the following components (in /umoles unless stated otherwise): potassium phosphate, p H 6.5, 100; ascorbate, 6.0; fumarate, 50; tyramine-/3/3'- H, 2.0, specific activity 1.15 X 10 CPM//umole; catalase, 300 units, dopamine j8-hydroxylase (4). Final volume, 0.68 ml. at 2 5 ° C . Octopamine determined by a minor modification of a published procedure (17). A 0.05 ml. sample of water was obtained by lyophilization and dissolved in 10 ml. of Bray's scintillation mixture. Radioactivity determined in a Packard liquid scintillation spectrometer; total counts collected were sufficient to yield a 5% coefficient of variation. The expected tritium release was calculated from the octopamine formed, assuming that the amount of tritium was the same in both /3 positions of the tyramine. The results for the amount of tritium released have teen corrected for the amount of exchangeable tritium initially present in the tyramine. a

3

5

T a b l e II.

R e a c t i o n o f Tyramine-£,/?'- H Dopamine /J-Hydroxylase

with

3

a

Experiment 1 2 3

4 5 6

Tritium

Enzyme Added, mg. 0 0.884 1.37 1.37 0.069 0.069

Ascorbate

(native) (boiled) (native) (boiled) (native)

+

+

Released

CPM 3.20 3.25 3.20 3.11 3.11 7.85

X X X X X X

10 10 10 10 10 10

3

3

3

3

3

5

0.18 0.18 0.18 0.17 0.17 43.0

• Complete reaction mixtures contained the following components (in /umoles): potassium phosphate, p H 6.5, 100; fumarate, 50; catalase, 300 units, tyramine, /3-/3'- H, 0.54, specific activity, 3.36 X 10 CPM/^mole. Ascorbate (6.0 /umoles) and dopamine 0-hydroxylase (4) were added where indicated. Final volume, 0.68 ml.; incubation time, 20 min. at 2 5 ° C . Reactions were stopped by freezing. A 0.1-ml. sample of water was obtained by lyophilization and dissolved in 10 ml. of Bray's scintillation mixture. Radioactivity determined in a Packard liquid scintillation spectrometer; total counts collected were sufficient to yield 5% coefficient of variation. 3

6

In Oxidation of Organic Compounds; Mayo, F.; Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

176

OXIDATION OF ORGANIC COMPOUNDS

Til

p l a c e ( E x p e r i m e n t 6), 4 3 % of t h e r a d i o a c t i v i t y w a s released i n t o t h e m e d i u m . S i n c e a 5% difference i n r a d i o a c t i v i t y b e t w e e n E x p e r i m e n t s 1 a n d 2 a n d E x p e r i m e n t s 3 a n d 4, c o u l d h a v e b e e n m e a s u r e d , these e x p e r i ­ ments w e r e sensitive e n o u g h to detect a p a r t i a l r e a c t i o n ( i n v o l v i n g sub­ strate a n d o x y g e n i n t h e absence of ascorbate) o c c u r r i n g at 0 . 0 1 % o f the c a t a l y t i c rate o b s e r v e d i n E x p e r i m e n t 6. If a ^ - s u b s t i t u t e d h y d r o p e r o x i d e of t h e substrate h a d b e e n f o r m e d i n a m o u n t s e q u a l to the a m o u n t of e n z y m e present, t h e e x p e c t e d a m o u n t of t r i t i u m released i n t h e presence of n a t i v e e n z y m e ( E x p e r i m e n t 4 ) s h o u l d h a v e e x c e e d e d that f o u n d i n its absence ( E x p e r i m e n t 3 ) b y a b o u t Downloaded by UNIV OF SYDNEY on September 4, 2014 | http://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0077.ch073

4000 C P M / 0 . 1 m l . T h e s e results s h o w that i t is u n l i k e l y that t h e h y d r o x y l a t i o n r e a c t i o n c a t a l y z e d b y d o p a m i n e ^ - h y d r o x y l a s e occurs as d e p i c t e d i n R e a c t i o n s 5 a n d 6. A ^ - s u b s t i t u t e d h y d r o p e r o x i d e of t h e substrate c o u l d s t i l l b e i n ­ v o l v e d i n t h e m e c h a n i s m i f t h e formation of t h e h y d r o p e r o x i d e r e q u i r e d the p a r t i c i p a t i o n of ascorbate.

Acknowledgment We

thank

C y r u s R . C r e v e l i n g f o r a generous

gift of t y r a m i n e

(-A/?'-) H. 3

Literature Cited

(1) Abbott, M. T., Schandl, E. K., Lee, R. F., Parker, T. S., Midgett, R. J., Biochim. Biophys. Acta 132, 525 (1967). (2) Bridgers, W. F., Kaufman, S., J. Biol. Chem. 237, 526 (1962). (3) Creveling, C. R., Daly, J. W., Witkop, B., Udenfriend, S., Biochim. Biophys. Acta 64, 125 (1962). (4) Friedman, S., Kaufman, S., J. Biol. Chem. 240, 4763 (1965). (5) Ibid., 241, 2256 (1966). (6) Goldstein, M., Contrera, J. F., J. Biol. Chem. 237, 1898 (1962). (7) Hausmann, E., Biochim. Biophys. Acta 133, 591 (1967). (8) Hutton, J. J., Tappel, A. L., Udenfriend, S., Biochem. Biophys. Res. Commun. 24, 179 (1966). (9) Katagiri, M., Takemori, S., Suzuki, K., Yasuda, H., J. Biol. Chem. 241, 5675 (1966). (10) Kaufman, S., J. Biol. Chem. 234, 2677 (1959). (11) Kaufman, S., "Oxygenases," O. Hayaishi, Ed., p. 129, Academic Press, New York, 1962. (12) Kaufman, S., Proc. Natl. Acad. Sci. 50, 1085 (1963). (13) Kivirikko, K. I., Prockop, D. J., Proc. Natl. Acad. Sci. 57, 782 (1967). (14) Levin, E. Y., Levenberg, B., Kaufman, S., J. Biol. Chem. 235, 2080 (1960). (15) Levin, E. Y., Kaufman, S., J. Biol. Chem. 236, 2043 (1961). (16) Mager, H. I. X., Berends, W., Biochim. Biophys. Acta 118, 440 (1966). (17) Pisano, J. J., Creveling, C. R., Udenfriend, S., Biochim. Biophys. Acta 43, 566 (1960). (18) Soloway, A. H., J. Theoret. Biol. 13, 100 (1966). RECEIVED

December 15, 1967.

In Oxidation of Organic Compounds; Mayo, F.; Advances in Chemistry; American Chemical Society: Washington, DC, 1968.