BIOCHEMICAL
Vol. 67, No. 2, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
STUDIES ON A POSSIBLE REACTION INTERMEDIATE OF II-HYDROXYPHENYLPYRUVATE DIOXYGENASE
Chieko
Nakai,
Mitsuhiro
Nozaki*
and Osamu Hayaishi
Department of Medical Chemistry Kyoto University Faculty of Medicine Kyoto, Japan Isao
Saito
and Teruo
Matsuura
Department of Synthetic Chemistry Kyoto University Faculty of Engineering Kyoto, Japan Received
September
17,1975
SUMiRY: A quinol has been proposed to be an intermediate of Howthe p-hydroxyphenylpyruvate dioxygenase-catalyzed reaction. ever, using a highly purified enzyme from bovine liver and chemicalno significant formation of homogentisate, ly synthesized quinol, from the quinol was observed the product of the enzymic reaction, under any conditions tested. Furthermore, the quinol showed no appreciable inhibition on the enzymic activity. In light of these findings, the reaction mechanism of the enzyme is discussed.
p-Hydroxyphenylpyruvate the
conversion
in which tion
diates
chain
(III)
direct In
fact,
* Present address: Medicine, Shiqa,
proposed
and migration
the enzyme in which
are involved
as interme(2) and further
1802 experiments
this
mechanism has been widely
evidence
to prove
another
mechanism
University
590
or disprove ((b)
(II),
hydroxyla-
with
Although
Shiga Japan.
namely,
by Goodwin and Witkop
et al. --
Q I9 75 by Academic Press, Inc. irz any form resewed.
of reproduction
mechanism for (IV)
catalyzes
to homogentisate
decarboxylation,
and a quinol
by Lindblad
(4),
(I)
are involved,
oxidative
A reaction
(a) in Scheme 1).
available.
reactions
ring,
was originally
accepted
Copyright All rights
of
(1).
peroxide
substantiated nism
types
of the benzene
a cyclic
(EC 1.13.11.27)
of p-hydroxyphenylpyruvate
three
of a side
dioxygenase
it
(3)
(Mecha-
has not
been
in Scheme 1) involving
of Medical
Science,
School
of
Vol. 67, No. 2, 1975
a peracid
BIOCHEMICAL
(V) as an intermediate
In previous intermediate tic
al.
the chemically
by Hamilton synthesized
(5). quinol
2-(l-hydroxy-4-oxo-2,5-cyclohexadien-l-yl)
nonenzymically
to
In order
an intermediate
RESEARCH COMMUNICATIONS
was proposed
in the same way as the O-acetate
(6).
ed with
experiments,
(IV),
acid,
converted et --
AND BIOPHYSICAL
to
of the
the highly
enzymic
describes
the
a free
intermediate.
(Z), was shown to be
in an alkaline
investigate
purified
report
(II)
whether
reaction,
against
solution or not
various
by Saito
the quinol
compound (IV)
enzyme under
evidence
ace-
is
was incubatThis
conditions.
participation
of a quinol
as
MATERIALS AND METHODS
p-Hydroxyphenylpyruvic DCIP-17 was a product ic
acid
(II)
of Merck
The quinol
oxygen
as described
grade,
were purchased
(IV)
previously
and urea
was synthesized All
and catalase
from
other
(I)
with
reagents,
sources
Kasei.
Homogentis
of Schwarz-Mann.
from Sigma,
(6).
from Tokyo
from singlet
analytical
and used without
purification.
liver
acetone et al. --
cedures
included
graphies tration
with with
(7) with gel
SP-Sephadex Sephadex
19 fold
enzyme thus
G-200.
Abbreviation:
with I~CIP,
with
buffer,
All
The purification
Bio-Gel
A-O.5
pH 5.6.
A-50,
a specific
activity
2,6-dichloroindophenol.
591
of
pro-
and gel
fil-
was carried
An overall
a yield
purification
22%. of
by
m, chromato-
the purification
with
from bovine
same method as described
C-50 and QAE-Sephadex
was achieved
obtained
the
was purified
some modifications.
filtration
4O in sodium acetate
of about
dioxygenase
powder by essentially
Lindblad
-l/
was obtained
from commercial
p-Hydroxyphenylpyruvate
at
(I)
and GSH were purchased
Boehringer.
further
acid
The purified
175 nmoles per
out
Vol. 67, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Mechanism
OH
(a)
OH
COO-
9-42 $=o
02,H+
Mechanism
Scheme 1. Proposed pyruvate dioxygenase. min per mg of protein
reaction
gel
some
modifications.
final
formation
volume
100 I.rg of
of
of
and the enzyme.
gentle
(II)
200 PM the
incubations
incubation, to
1,4-thiazine
and the
was not
ethanol
with
faint
out
at 37' (II)
absorbance
conditions,
for
formed at
in a
buffer,
pH 7.5,
10 @ DCIP,
1 mM_GSH,
addition
at 37'
for
of
by the addition
6 g glycine-NaOH 60 min.
was completely
of buffer,
During
conversion
the
15 min with
this converted
390 nm (E = 5,520)
nonenzymic
(8) with
contained
by the
the
of
was meas(IV)
to
observed.
Compound (II) graphy
(I),
and 100 ~1 of
homogentisate
Under these
ured. (II)
the
substrate
was terminated
incubated
et al. --
phosphate
was started
The reaction
and further
several
by measuring
assay mixture
were carried
100 ~1 of 0.4 M_L-cysteine pH 10.6,
band with
routinely
150 mM_potassium
The reaction
shaking.
p-hydroxyphenyl-
by the method of Fellman
The standard
catalase,
enzyme and the
was assayed
1.0 ml,
thick
for
electrophoresis.
The enzyme activity of
mechanisms
showed a major
bands on acrylamide
rate
( b)
was also
a solvent
(100:3:5,
system
V/V/V)
detected of ethyl
either
by thin
acetate,
acetic
or by homogentisicase
which
592
layer acid
chromatoand
specifically
Vol.
67, No.
2, 1975
BIOCHEMICAL
Table from
I.
Enzymic
AND
BIOPHYSICAL
formation
of
p-hydroxyphenylpyruvate
RESEARCH
homogentisate (I)
(II)
and quinol
Homogentisate (nmoles/l5
Additions
COMMUNICATIONS
(IV)
formed min)
p-hydroxyphenylpyruvate
quinol
None
3.3
0.4
DCIP
6.0
0.0
GSH
12.3
0.0
Asc
41.6
0.0
GSH + Asc
61.4
0.0
DCIP
+ GSH
80.3
0.6
DCIP
+ ASC
72.1
0.0
DCIP
+ GSH + Asc
80.3
0.2
The reaction mixture contained in a final volume of 1.0 ml, 150 mM potassium phosphate buffer, pH 7.5, 100 Fig of catalase, 200 @ eiFher p-hydroxyphenylpyruvate (I) or the quinol (IV), and 30 pg of the enzyme. Where indicated, the following were added: 10 CIFJ DCIP, 1 mM GSH, 1 mM ascorbic acid (Asc). Other conditions were the same as descrTbed under Materials and Methods. The values less than 1.0 are not significant with this assay method.
cleaves
the
an absorption
benzene
ring
maximum
of
at
(II)
320
to
form
having
maleylacetoacetate
nm.
RESULTS AND DISCUSSION
As enzyme
shown showed
The activity
in
Table
a little was
I,
when
(I)
activity
augmented
in
by the
593
was
used
the
absence
addition
as
of
the of
substrate, added
various
cofactors.
cofactors
the
Vol. 67, No. 2, 1975
BIOCHEMICAL
and maximum activity bination (IV)
with
was obtained
GSH and/or
was used as the
was observed reported
either
amount at pH 12 at
24" within
experiments
increasing
the quinol
the
range
from mixture.
original
activity
respectively,
the
phenylpyruvate
(I)
These results
be released
formed. fact
Since
that
general these
reacting
applicability results
studies
Mechanism
are required
to
86,
various
1.0,
from
the pH over 4.0 M) to the
However,
(IV)
with
even when the p-hydroxyto 20 @.
disprove but
Mechanism
indicate
until
that
the end product
10) and -p-fluorophenylpyruvate
the para-hydroxy
for may
has been
group,
the (9)
the enzyme weakens the case for (a).
Needless
nor exclude
Mechanism
to clarify
(a)
quinol
(9,
prove
Fur-
1 mM - quinol
requires
of
in no
observed.
(a)
substrates
150 l.iq,
2.0 and 4.0 g urea,
was decreased
necessarily
other
62 and 10% of its
was observed
of Mechanism
neither
experiments
under
(0.5
100,
(II)
the ES-complex
As
stoichiometric
assay condition.
dioxygenase
phenylpyruvate
are slowly
urea
of 0.5,
concentration
from
out
assay conditions,
p-hydroxyphenylpyruvate not
or adding
of
(II)
cofactors.
to 1 mM, varying
standard
do not
of
the amount of enzyme to
inhibition
standard
of
when
hours.
carried
in the presence
no significant
under
several
formation
hand,
used in these
The enzyme retained
the
DCIP in com-
formation
in an almost
concentration
case was the enzymic
(IV)
(II)
were also
to 9.0,
under
thermore,
to
increasing
5.6
reaction
no significant
compound (IV)
converted
of
On the other
absence or presence
was nonenzymically
including
acid.
substrate,
(6),
conditions
in the presence
ascorbic
in the
previously
Similar
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
reaction
to say, (b).
however,
Further
mechanism of the
enzyme. The quinol in the
reaction
(IV)
has also
catalyzed
been postulated
by p-hydroxyphenylacetate -
594
as an intermediate 1-hydroxylase,
BIOCHEMICAL
Vol. 67, No. 2, 1975
an enzyme which to homogentisate Since
(11). required
catalyzes (II)
conversion
in the presence
the para-hydroxygroup
for
this
enzymic
seems more probable. have so far
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
not been carried
of 02 and a reducing
in the
substrate
is
agent
apparently
quinol
(IV)
as an intermediate
experiments
with
this
reaction,
However,
of p-hydroxyphenylacetate -
hydroxylase
out.
ACKNOWLEDGMENTS: This work has been supported in part by grants from the Japan Foundation for Applied Enzymology, the Medical Research Foundation for Geriatric Diseases, and from the Scientific Research Fund of the Ministry of Education of Japan. We are indebted to Drs. Gordon A. Hamilton, Bernhard Witkop and Shozo Yamamoto for their valuable discussions and critical readings, and to Dr. Clark M. Edson for his aid in the preparation of this manuscript. REFERENCES 1. 2. 3. 4.
5.
6. 7. 8. 9. 10. 11.
J. Biol. Hager, S. E., Gregerman, R. I. and Knox, W. E. (1957) Chem. -'225 935-947. Goodwin, S. and Witkop, B. (1957) J. Amer. Chem. Sot. 79, 179-185. Lindblad, B., Lindstedt, G. and Lindstedt, S. (1970) J. Amer. Chem. Sot. 92, 7446-7449. S. (1974) in Molecular MechaAbbott, M. T. and Udenfriend, nisms of Oxygen Activation (Hayaishi, 0. ed.), pp. 167-214, Academic Press, New York. Hamilton, G. A. (1971) in Progress in Bioorganic Chemistry (Kaiser, E. T. and Kezdy, F. J. eds.) Vol. I, pp. 83-157, Wiley, New York. Saito, I., Yamane, M., Shimazu, H., Matsuura, T. and Cahnmann, H. J. (1975) Tetrahedron Lett. 2, 641-644. Lindblad, B., Lindstedt, S., Olander, B. and Omfeldt, Acta Chem. Stand. 25, 329-330. Fellman, J. H., Fujita, T. S. and Roth, E. S. (1972) Biophys. Acta 284, 90-100.
M. (1971)
Taniguchi, Chem. z,
J.
K., Kappe, 3389-3395.
T. and Armstrong,
M. D.
(1964)
Biochim. Biol.
Biochim. Fellman, J. H., Fujita, T. S. and Roth, E. S. (1972) Acta 268, 601-604. Biophys. Hareland, W. A., Crawford, R. L., Chapman, P. J. and Dagley, S. (1975) J. Bacterial. 121, 272-285.
595
Vol. 67, No. 2, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
STUDIES ON A POSSIBLE REACTION INTERMEDIATE OF II-HYDROXYPHENYLPYRUVATE DIOXYGENASE
Chieko
Nakai,
Mitsuhiro
Nozaki*
and Osamu Hayaishi
Department of Medical Chemistry Kyoto University Faculty of Medicine Kyoto, Japan Isao
Saito
and Teruo
Matsuura
Department of Synthetic Chemistry Kyoto University Faculty of Engineering Kyoto, Japan Received
September
17,1975
SUMiRY: A quinol has been proposed to be an intermediate of Howthe p-hydroxyphenylpyruvate dioxygenase-catalyzed reaction. ever, using a highly purified enzyme from bovine liver and chemicalno significant formation of homogentisate, ly synthesized quinol, from the quinol was observed the product of the enzymic reaction, under any conditions tested. Furthermore, the quinol showed no appreciable inhibition on the enzymic activity. In light of these findings, the reaction mechanism of the enzyme is discussed.
p-Hydroxyphenylpyruvate the
conversion
in which tion
diates
chain
(III)
direct In
fact,
* Present address: Medicine, Shiqa,
proposed
and migration
the enzyme in which
are involved
as interme(2) and further
1802 experiments
this
mechanism has been widely
evidence
to prove
another
mechanism
University
590
or disprove ((b)
(II),
hydroxyla-
with
Although
Shiga Japan.
namely,
by Goodwin and Witkop
et al. --
Q I9 75 by Academic Press, Inc. irz any form resewed.
of reproduction
mechanism for (IV)
catalyzes
to homogentisate
decarboxylation,
and a quinol
by Lindblad
(4),
(I)
are involved,
oxidative
A reaction
(a) in Scheme 1).
available.
reactions
ring,
was originally
accepted
Copyright All rights
of
(1).
peroxide
substantiated nism
types
of the benzene
a cyclic
(EC 1.13.11.27)
of p-hydroxyphenylpyruvate
three
of a side
dioxygenase
it
(3)
(Mecha-
has not
been
in Scheme 1) involving
of Medical
Science,
School
of
Vol. 67, No. 2, 1975
a peracid
BIOCHEMICAL
(V) as an intermediate
In previous intermediate tic
al.
the chemically
by Hamilton synthesized
(5). quinol
2-(l-hydroxy-4-oxo-2,5-cyclohexadien-l-yl)
nonenzymically
to
In order
an intermediate
RESEARCH COMMUNICATIONS
was proposed
in the same way as the O-acetate
(6).
ed with
experiments,
(IV),
acid,
converted et --
AND BIOPHYSICAL
to
of the
the highly
enzymic
describes
the
a free
intermediate.
(Z), was shown to be
in an alkaline
investigate
purified
report
(II)
whether
reaction,
against
solution or not
various
by Saito
the quinol
compound (IV)
enzyme under
evidence
ace-
is
was incubatThis
conditions.
participation
of a quinol
as
MATERIALS AND METHODS
p-Hydroxyphenylpyruvic DCIP-17 was a product ic
acid
(II)
of Merck
The quinol
oxygen
as described
grade,
were purchased
(IV)
previously
and urea
was synthesized All
and catalase
from
other
(I)
with
reagents,
sources
Kasei.
Homogentis
of Schwarz-Mann.
from Sigma,
(6).
from Tokyo
from singlet
analytical
and used without
purification.
liver
acetone et al. --
cedures
included
graphies tration
with with
(7) with gel
SP-Sephadex Sephadex
19 fold
enzyme thus
G-200.
Abbreviation:
with I~CIP,
with
buffer,
All
The purification
Bio-Gel
A-O.5
pH 5.6.
A-50,
a specific
activity
2,6-dichloroindophenol.
591
of
pro-
and gel
fil-
was carried
An overall
a yield
purification
22%. of
by
m, chromato-
the purification
with
from bovine
same method as described
C-50 and QAE-Sephadex
was achieved
obtained
the
was purified
some modifications.
filtration
4O in sodium acetate
of about
dioxygenase
powder by essentially
Lindblad
-l/
was obtained
from commercial
p-Hydroxyphenylpyruvate
at
(I)
and GSH were purchased
Boehringer.
further
acid
The purified
175 nmoles per
out
Vol. 67, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Mechanism
OH
(a)
OH
COO-
9-42 $=o
02,H+
Mechanism
Scheme 1. Proposed pyruvate dioxygenase. min per mg of protein
reaction
gel
some
modifications.
final
formation
volume
100 I.rg of
of
of
and the enzyme.
gentle
(II)
200 PM the
incubations
incubation, to
1,4-thiazine
and the
was not
ethanol
with
faint
out
at 37' (II)
absorbance
conditions,
for
formed at
in a
buffer,
pH 7.5,
10 @ DCIP,
1 mM_GSH,
addition
at 37'
for
of
by the addition
6 g glycine-NaOH 60 min.
was completely
of buffer,
During
conversion
the
15 min with
this converted
390 nm (E = 5,520)
nonenzymic
(8) with
contained
by the
the
of
was meas(IV)
to
observed.
Compound (II) graphy
(I),
and 100 ~1 of
homogentisate
Under these
ured. (II)
the
substrate
was terminated
incubated
et al. --
phosphate
was started
The reaction
and further
several
by measuring
assay mixture
were carried
100 ~1 of 0.4 M_L-cysteine pH 10.6,
band with
routinely
150 mM_potassium
The reaction
shaking.
p-hydroxyphenyl-
by the method of Fellman
The standard
catalase,
enzyme and the
was assayed
1.0 ml,
thick
for
electrophoresis.
The enzyme activity of
mechanisms
showed a major
bands on acrylamide
rate
( b)
was also
a solvent
(100:3:5,
system
V/V/V)
detected of ethyl
either
by thin
acetate,
acetic
or by homogentisicase
which
592
layer acid
chromatoand
specifically
Vol.
67, No.
2, 1975
BIOCHEMICAL
Table from
I.
Enzymic
AND
BIOPHYSICAL
formation
of
p-hydroxyphenylpyruvate
RESEARCH
homogentisate (I)
(II)
and quinol
Homogentisate (nmoles/l5
Additions
COMMUNICATIONS
(IV)
formed min)
p-hydroxyphenylpyruvate
quinol
None
3.3
0.4
DCIP
6.0
0.0
GSH
12.3
0.0
Asc
41.6
0.0
GSH + Asc
61.4
0.0
DCIP
+ GSH
80.3
0.6
DCIP
+ ASC
72.1
0.0
DCIP
+ GSH + Asc
80.3
0.2
The reaction mixture contained in a final volume of 1.0 ml, 150 mM potassium phosphate buffer, pH 7.5, 100 Fig of catalase, 200 @ eiFher p-hydroxyphenylpyruvate (I) or the quinol (IV), and 30 pg of the enzyme. Where indicated, the following were added: 10 CIFJ DCIP, 1 mM GSH, 1 mM ascorbic acid (Asc). Other conditions were the same as descrTbed under Materials and Methods. The values less than 1.0 are not significant with this assay method.
cleaves
the
an absorption
benzene
ring
maximum
of
at
(II)
320
to
form
having
maleylacetoacetate
nm.
RESULTS AND DISCUSSION
As enzyme
shown showed
The activity
in
Table
a little was
I,
when
(I)
activity
augmented
in
by the
593
was
used
the
absence
addition
as
of
the of
substrate, added
various
cofactors.
cofactors
the
Vol. 67, No. 2, 1975
BIOCHEMICAL
and maximum activity bination (IV)
with
was obtained
GSH and/or
was used as the
was observed reported
either
amount at pH 12 at
24" within
experiments
increasing
the quinol
the
range
from mixture.
original
activity
respectively,
the
phenylpyruvate
(I)
These results
be released
formed. fact
Since
that
general these
reacting
applicability results
studies
Mechanism
are required
to
86,
various
1.0,
from
the pH over 4.0 M) to the
However,
(IV)
with
even when the p-hydroxyto 20 @.
disprove but
Mechanism
indicate
until
that
the end product
10) and -p-fluorophenylpyruvate
the para-hydroxy
for may
has been
group,
the (9)
the enzyme weakens the case for (a).
Needless
nor exclude
Mechanism
to clarify
(a)
quinol
(9,
prove
Fur-
1 mM - quinol
requires
of
in no
observed.
(a)
substrates
150 l.iq,
2.0 and 4.0 g urea,
was decreased
necessarily
other
62 and 10% of its
was observed
of Mechanism
neither
experiments
under
(0.5
100,
(II)
the ES-complex
As
stoichiometric
assay condition.
dioxygenase
phenylpyruvate
are slowly
urea
of 0.5,
concentration
from
out
assay conditions,
p-hydroxyphenylpyruvate not
or adding
of
(II)
cofactors.
to 1 mM, varying
standard
do not
of
the amount of enzyme to
inhibition
standard
of
when
hours.
carried
in the presence
no significant
under
several
formation
hand,
used in these
The enzyme retained
the
DCIP in com-
formation
in an almost
concentration
case was the enzymic
(IV)
(II)
were also
to 9.0,
under
thermore,
to
increasing
5.6
reaction
no significant
compound (IV)
converted
of
On the other
absence or presence
was nonenzymically
including
acid.
substrate,
(6),
conditions
in the presence
ascorbic
in the
previously
Similar
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
reaction
to say, (b).
however,
Further
mechanism of the
enzyme. The quinol in the
reaction
(IV)
has also
catalyzed
been postulated
by p-hydroxyphenylacetate -
594
as an intermediate 1-hydroxylase,
BIOCHEMICAL
Vol. 67, No. 2, 1975
an enzyme which to homogentisate Since
(11). required
catalyzes (II)
conversion
in the presence
the para-hydroxygroup
for
this
enzymic
seems more probable. have so far
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
not been carried
of 02 and a reducing
in the
substrate
is
agent
apparently
quinol
(IV)
as an intermediate
experiments
with
this
reaction,
However,
of p-hydroxyphenylacetate -
hydroxylase
out.
ACKNOWLEDGMENTS: This work has been supported in part by grants from the Japan Foundation for Applied Enzymology, the Medical Research Foundation for Geriatric Diseases, and from the Scientific Research Fund of the Ministry of Education of Japan. We are indebted to Drs. Gordon A. Hamilton, Bernhard Witkop and Shozo Yamamoto for their valuable discussions and critical readings, and to Dr. Clark M. Edson for his aid in the preparation of this manuscript. REFERENCES 1. 2. 3. 4.
5.
6. 7. 8. 9. 10. 11.
J. Biol. Hager, S. E., Gregerman, R. I. and Knox, W. E. (1957) Chem. -'225 935-947. Goodwin, S. and Witkop, B. (1957) J. Amer. Chem. Sot. 79, 179-185. Lindblad, B., Lindstedt, G. and Lindstedt, S. (1970) J. Amer. Chem. Sot. 92, 7446-7449. S. (1974) in Molecular MechaAbbott, M. T. and Udenfriend, nisms of Oxygen Activation (Hayaishi, 0. ed.), pp. 167-214, Academic Press, New York. Hamilton, G. A. (1971) in Progress in Bioorganic Chemistry (Kaiser, E. T. and Kezdy, F. J. eds.) Vol. I, pp. 83-157, Wiley, New York. Saito, I., Yamane, M., Shimazu, H., Matsuura, T. and Cahnmann, H. J. (1975) Tetrahedron Lett. 2, 641-644. Lindblad, B., Lindstedt, S., Olander, B. and Omfeldt, Acta Chem. Stand. 25, 329-330. Fellman, J. H., Fujita, T. S. and Roth, E. S. (1972) Biophys. Acta 284, 90-100.
M. (1971)
Taniguchi, Chem. z,
J.
K., Kappe, 3389-3395.
T. and Armstrong,
M. D.
(1964)
Biochim. Biol.
Biochim. Fellman, J. H., Fujita, T. S. and Roth, E. S. (1972) Acta 268, 601-604. Biophys. Hareland, W. A., Crawford, R. L., Chapman, P. J. and Dagley, S. (1975) J. Bacterial. 121, 272-285.
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