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.
