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The participation of copper in tryptophan pyrrolase action
Vol. 2 1, No. 4, 1965
THE
BIOCHEMICAL
PARTICIPATION
OF
COPPER
Hiroo Departments
of
Ret eived
The
8,
the
uncler valence
of
proposed:
(a)
Tokuyama
and
process
(Feigelson
the:
steady
may
heme
bound is
enzyme
the
in
the
and
Surgeons
(b)
the
undergeo 1964,
herein
oscillation
in
valence
by
( c ) only
cyclic
which
tryptophan virtue
sensitivity
the
presence
catalytic
of
copper
process
to
active
finding,
of the
which
hematin of
catalytic
catalytic
during
participation
pyrrolase
of the
of
the
is
reduction
role
this
the
form
A new
the
but
during
divalent
been
1959;
trivalent,
and
and
have
Knox,
state
1964).
process in
and is
oxidation
(Hayaishi,
reported,
the
the
process,
(Tanaka form
catalytic
microbial
active
been
concerning
catalytic
heme
reaction the
the
active
1965);
has
hypotheses
during is
a hemoprotein,
formylkynurenine
alternative
undergoes
suggested the
ACTION+
of Physicians N. Y.
pyrrolase, to
form
1964);
clarify
copper
and
divalent
catalytic
is
College York,
New
L-tryptophan
coenzyme
coenzyme
therein
strongly
heme
et-- al.
state
PYRROLASE
Feigelson””
tryptophan
Three
may
ultimately
cofactor
which of
Knox,
coenzyme
the
the
only
he:natin
bu:
by
investigation.
state
Philip
and Medicine, University,
oxygenation
active
and
TRYPTOPHAN
1965
mechanism
catalyzes
IN :k
Maeno
Biochemistry Columbia
October
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
enzyme
reaction in
specific
the
purified chelating
agents. Pseudomonas
ATCC
rI’his project supported #Science Foundation ( G * Visiting Scientist from Osaka, Japan. ;(::* Career Investigator of (I- 104).
11299
was
cultivated
in
the
presence
of
0. i ‘%
in part by research grants from the National 20878) and from the U.S. P. H. S. (CA-02332). the Department of Biochemistry, Osaka University, the
Health
Research
297
Council
of the
City
of New
York
Vol. 21, No. 4, 1965
BIOCHEMICAL
L-tryptophan.
The
previously
described
by zone tion
enzyme
10m3M
and Feigelson,
1962).
purified
After
column
elution,
ammonium
sulfate,
and finally
subjected
using
starch
Under
these
single
brown
band
with
the enzyme
and protein
reported
(Feigelson
et al. --
of one pmole
Poillon
and Dawson The
grams
was
the enzyme increase
with
respect wherein
was
recovered
The
copper
proceeded.
noted
that when
much
cuproine
at by adding
at 7.5 volts/cm.,
Verona1
towards
buffer.
the anode
the excised
as a
starch
Tryptophan
were
measured
unit
is defined
per
enzyme
is presented
to copper
ment
of formylkynurenine using
elution
pH 7. 0.
One enzyme
of a typical
in purity.
as the purification
1965).
further
16 hours
from
buffer,
and then
( p = 0. 1, pH 8. 3)
in the same
eluted
bacteria,
minute
as previously as catalytic
at 25’C.
according
Total
to the method
of
(1963).
bacteria activity
for
concentrations
determined
summary
of wet
was
induced
concentrated
buffer
moves
phosphate
activity
content
Verona1
purifica-
(Greengard
gradient
was
medium
enzyme
potassium
copper
using
the holoenzyme The
from
as
purification
in the enzyme
sulfate,
electrophoresis
as supporting
a further
extracted
solution
against
pyrrolase
formation
was
out essentially
the enzyme
and ammonium
to zone
band.
with used
chromatography
conditions,
10S2M
1965)
solutions
enzyme
dialyzed
grains
carried
to stabilize
streptomycin
by DEAE
pH 7. 0.
the
L-tryptophan The
was
et -- al.
All
contained
with
purification
(Feigelson
electrophoresis.
fractionated
AND BIOPHYSICAL RESEARCH COMMUNKATIONS
in Table with
I.
was further
298
was purified
10
percent
of
seventy-fold enzyme
of the tryptophan
evident protein
with
Twenty-nine
of the purified
Enrichment
contaminating
starting
an approximately
content
is particularly
the enzyme
purification
following removed. 2. 6 fold
increased pyrrolase
DFAE
treat-
It may by zone
be
fraction
sulfate
1. 3
1.0
DEAE
Electrophoresis
4. 5
54.0
Streptomycin
Ammonium
45.5
Volume ml.
Extract
Preparation
Purification
Copper
10.0
35.0
77.0
29.0
50.0
Protein mg. /ml.
and
1. 36
1.67
/ml.
22.
30.0 0
12. 6
E. U.
Content
I.
2.20
0.857
0. 164
0.047
0.033
Activity E. U. /mg. pro t ein
of Pseudomonas
Table
29
51
75
97
100
Recovery
Tryptophan
30
38
108
144
114
m~mole/ml.
Pyrrolase Copper m)*mole/mg.
10.8
4. ii
1.48
1.03
0. 76
protein
Vol. 21, No. 4, 1965
BIOCHEMICAL
electrophoresis,
the
indicating
a constant
content be
of
such by
Richmond,
is
bound
considered
an
To inhibitor agents.
As
tryptophan
integral
1955),
1932),
chelate salt
markedly
inhibited
show
which
any
the
enzyme.
been oxidase
(Okunuki,
1962).
catalyzing
the
both
a dissociable
and
copper
of
this
its
copper
reported
have that
enzyme
of
hematin been
at
existing will
Gut
component.
addition
copper
and
and
should
which the
copper
be
into
all disulfonated
such
a( , 4’
- dipyridyl,
all as
and
EDTA,
did
concentrations. to heme is
iron,
inhibited is
organic
the
and
by
10s2
the
only
M
expansion knowledge
salicylaldoxime
oxygenase, in
Feigelson,
catalytic the
our
mammalian
molecule,
concerning
300
which
agents,
in
in
Burnett,
chelating
to
stage
1955)
and
1958),
(Greengard
and
chelating
Smith,
an
partake
copper,
imonsen
and
coenzyme
this
that
of bathocuproine
pyrrolase
revision At
suggest
copper
Bollier,
(Diehl
in
hypotheses require
Co.,
copper
bound
10 -3M)(S’
comparable
oxygen
shown
not
(10m2M)
levels
tryptophan
insertion
(Bio-Rad
enzyme
and
Other
that,
However,
chelex-100
specific
iOT4M to
contains
could
findings
(4 x
effect
copper
preparations
extraneous
of the
(Peterson
specific
The
diethyldithiocarbamate
and
inhibitory
cytochrome
evident
role
II,
is
activity.
molecule
o-phenanthroline,
It has
is
removing
employing
Table
Cut+,
8-hydroxyquinoline, not
of
2. 6 fold,
structure.
functional
(4 x 10s3M)
disodium
with
salicylaldoxime
cuprizone
specifically
enzyme
These
of its
initiated
in
enzyme
pyrrolase
part
were
increased
pyrrolase
a protein.
the
also
to
capable
to the
shown
(McFarlane,
is
to
studies
copper
of the
bound
determine
was
tryptophan
) which
loosely
tightly
of
treatment
Calif.
be
content
ratio
purified
decreased
may
copper
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
which 1962)
process. catalytic
It
mechanism
to include one
a role may
suspect
for
Vol. 21, No. 4, 1965
BIOCHEMICAL
AND BIOPHYSICAL
‘Table Inhibition
of Tryptophan
Inhibitors
RESEARCH COMMUNICATIONS
II.
Pyrrolase
Activity
By Chelating
Agents
Concentrations
Inhibition
8 -Hydroxyquinoline
1 x 10-2
0
d , d’-dipyridyl
1 x 10-Z
0
E,DTA
1 x 10 -2
11
1 x 10 -3
11
1 x 10-s 1 x 10-4
100
Sodium
Azide
E:CN
(%)
80
1)iethyldithiocarbamate
1 x 10 -2 1 x 10 -3
68 12
S alicylaldoxime
8 4
x 1O-3 x 10-8 1 x 10-3
86 62 27
(Zuprizone
4
x 1o-3
52
13athocuproinedisulfonate
9 x 10-4 3 x 10-4 9 x 10-5
100 39 22
The enzyme was preincubated with chelating agents for 10 minutes ;tnd then L-tryptophan, hematin and ascorbate +ere added. The rate of ::ormylkynurenine formation was then measured spectrophotometrically as previously described (Fei.gelson et Cuprizone, being water -- al., 1965). :.nsoluble, was dissolved into 50% ethanol; in this control experiment, the same volume of 50% ethanol was added to the incubation mixture.
that
the substrates
are
bound
enzyme. between, pyrrolase
of tryptophan
pyrrolase,
oxygen
and tryptophan,
respectively
to the cuprous
and hematin
The
catalytic
of, and the possible
the are
specific enzyme
bound
under
further
copper
roles
and hematin
investigation.
301
components
moities
of the
interaction in tryptophan
Vol. 21, No. 4, 1965
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES Reagents Cuproine, Neocuproine, Diehl, H. and Smith G. F., “The Copper Bathocuproine” G. F. Smith Co., Columbus, Ohio (1958). Feigelson, P., Ishimura, Y., and Hayaishi, O., Biochem. Biophys. Res. Commun. 14:96 (1964). Feigelson, P., Ishimura Y., and Hayaishi, 0.) Biochim. Biophys. Acta, 95 283 (1965). P., J. Biol. Chem., 237, 1903 (1962). Greengard, 0.) and Feigelson, Congress of Biochemxtry (New York) Hayaishi, O., Sixth International -1.u. B. vol. 33, p. 31 (1964). McFarlane, W., Biochem. J., 24, 1022 (1932). edited by 0. Hayaishi, Academic Press Okunuki, K., in “Oxygenases” New York and London, p/409 (1962). Peterson, R., and Bollier, M., Anal. Chem., 27; 1195 (1955). Poillon, N. W., and Dawson, C.H., Biochim. Bgphys. Acta, 72, 27 (1963). Simonsen, S. H., and Burnett, H. M., Anal. Chem., 22; 1336 (1955). W. E., J. Biol. Chem., 234, 1162 (1959). Tanaka, T., and Knox, Biophys. Acta, 81, 201 (1964). Tokuyama, K., and Knox, W. E., Biochim.
302
THE
BIOCHEMICAL
PARTICIPATION
OF
COPPER
Hiroo Departments
of
Ret eived
The
8,
the
uncler valence
of
proposed:
(a)
Tokuyama
and
process
(Feigelson
the:
steady
may
heme
bound is
enzyme
the
in
the
and
Surgeons
(b)
the
undergeo 1964,
herein
oscillation
in
valence
by
( c ) only
cyclic
which
tryptophan virtue
sensitivity
the
presence
catalytic
of
copper
process
to
active
finding,
of the
which
hematin of
catalytic
catalytic
during
participation
pyrrolase
of the
of
the
is
reduction
role
this
the
form
A new
the
but
during
divalent
been
1959;
trivalent,
and
and
have
Knox,
state
1964).
process in
and is
oxidation
(Hayaishi,
reported,
the
the
process,
(Tanaka form
catalytic
microbial
active
been
concerning
catalytic
heme
reaction the
the
active
1965);
has
hypotheses
during is
a hemoprotein,
formylkynurenine
alternative
undergoes
suggested the
ACTION+
of Physicians N. Y.
pyrrolase, to
form
1964);
clarify
copper
and
divalent
catalytic
is
College York,
New
L-tryptophan
coenzyme
coenzyme
therein
strongly
heme
et-- al.
state
PYRROLASE
Feigelson””
tryptophan
Three
may
ultimately
cofactor
which of
Knox,
coenzyme
the
the
only
he:natin
bu:
by
investigation.
state
Philip
and Medicine, University,
oxygenation
active
and
TRYPTOPHAN
1965
mechanism
catalyzes
IN :k
Maeno
Biochemistry Columbia
October
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
enzyme
reaction in
specific
the
purified chelating
agents. Pseudomonas
ATCC
rI’his project supported #Science Foundation ( G * Visiting Scientist from Osaka, Japan. ;(::* Career Investigator of (I- 104).
11299
was
cultivated
in
the
presence
of
0. i ‘%
in part by research grants from the National 20878) and from the U.S. P. H. S. (CA-02332). the Department of Biochemistry, Osaka University, the
Health
Research
297
Council
of the
City
of New
York
Vol. 21, No. 4, 1965
BIOCHEMICAL
L-tryptophan.
The
previously
described
by zone tion
enzyme
10m3M
and Feigelson,
1962).
purified
After
column
elution,
ammonium
sulfate,
and finally
subjected
using
starch
Under
these
single
brown
band
with
the enzyme
and protein
reported
(Feigelson
et al. --
of one pmole
Poillon
and Dawson The
grams
was
the enzyme increase
with
respect wherein
was
recovered
The
copper
proceeded.
noted
that when
much
cuproine
at by adding
at 7.5 volts/cm.,
Verona1
towards
buffer.
the anode
the excised
as a
starch
Tryptophan
were
measured
unit
is defined
per
enzyme
is presented
to copper
ment
of formylkynurenine using
elution
pH 7. 0.
One enzyme
of a typical
in purity.
as the purification
1965).
further
16 hours
from
buffer,
and then
( p = 0. 1, pH 8. 3)
in the same
eluted
bacteria,
minute
as previously as catalytic
at 25’C.
according
Total
to the method
of
(1963).
bacteria activity
for
concentrations
determined
summary
of wet
was
induced
concentrated
buffer
moves
phosphate
activity
content
Verona1
purifica-
(Greengard
gradient
was
medium
enzyme
potassium
copper
using
the holoenzyme The
from
as
purification
in the enzyme
sulfate,
electrophoresis
as supporting
a further
extracted
solution
against
pyrrolase
formation
was
out essentially
the enzyme
and ammonium
to zone
band.
with used
chromatography
conditions,
10S2M
1965)
solutions
enzyme
dialyzed
grains
carried
to stabilize
streptomycin
by DEAE
pH 7. 0.
the
L-tryptophan The
was
et -- al.
All
contained
with
purification
(Feigelson
electrophoresis.
fractionated
AND BIOPHYSICAL RESEARCH COMMUNKATIONS
in Table with
I.
was further
298
was purified
10
percent
of
seventy-fold enzyme
of the tryptophan
evident protein
with
Twenty-nine
of the purified
Enrichment
contaminating
starting
an approximately
content
is particularly
the enzyme
purification
following removed. 2. 6 fold
increased pyrrolase
DFAE
treat-
It may by zone
be
fraction
sulfate
1. 3
1.0
DEAE
Electrophoresis
4. 5
54.0
Streptomycin
Ammonium
45.5
Volume ml.
Extract
Preparation
Purification
Copper
10.0
35.0
77.0
29.0
50.0
Protein mg. /ml.
and
1. 36
1.67
/ml.
22.
30.0 0
12. 6
E. U.
Content
I.
2.20
0.857
0. 164
0.047
0.033
Activity E. U. /mg. pro t ein
of Pseudomonas
Table
29
51
75
97
100
Recovery
Tryptophan
30
38
108
144
114
m~mole/ml.
Pyrrolase Copper m)*mole/mg.
10.8
4. ii
1.48
1.03
0. 76
protein
Vol. 21, No. 4, 1965
BIOCHEMICAL
electrophoresis,
the
indicating
a constant
content be
of
such by
Richmond,
is
bound
considered
an
To inhibitor agents.
As
tryptophan
integral
1955),
1932),
chelate salt
markedly
inhibited
show
which
any
the
enzyme.
been oxidase
(Okunuki,
1962).
catalyzing
the
both
a dissociable
and
copper
of
this
its
copper
reported
have that
enzyme
of
hematin been
at
existing will
Gut
component.
addition
copper
and
and
should
which the
copper
be
into
all disulfonated
such
a( , 4’
- dipyridyl,
all as
and
EDTA,
did
concentrations. to heme is
iron,
inhibited is
organic
the
and
by
10s2
the
only
M
expansion knowledge
salicylaldoxime
oxygenase, in
Feigelson,
catalytic the
our
mammalian
molecule,
concerning
300
which
agents,
in
in
Burnett,
chelating
to
stage
1955)
and
1958),
(Greengard
and
chelating
Smith,
an
partake
copper,
imonsen
and
coenzyme
this
that
of bathocuproine
pyrrolase
revision At
suggest
copper
Bollier,
(Diehl
in
hypotheses require
Co.,
copper
bound
10 -3M)(S’
comparable
oxygen
shown
not
(10m2M)
levels
tryptophan
insertion
(Bio-Rad
enzyme
and
Other
that,
However,
chelex-100
specific
iOT4M to
contains
could
findings
(4 x
effect
copper
preparations
extraneous
of the
(Peterson
specific
The
diethyldithiocarbamate
and
inhibitory
cytochrome
evident
role
II,
is
activity.
molecule
o-phenanthroline,
It has
is
removing
employing
Table
Cut+,
8-hydroxyquinoline, not
of
2. 6 fold,
structure.
functional
(4 x 10s3M)
disodium
with
salicylaldoxime
cuprizone
specifically
enzyme
These
of its
initiated
in
enzyme
pyrrolase
part
were
increased
pyrrolase
a protein.
the
also
to
capable
to the
shown
(McFarlane,
is
to
studies
copper
of the
bound
determine
was
tryptophan
) which
loosely
tightly
of
treatment
Calif.
be
content
ratio
purified
decreased
may
copper
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
which 1962)
process. catalytic
It
mechanism
to include one
a role may
suspect
for
Vol. 21, No. 4, 1965
BIOCHEMICAL
AND BIOPHYSICAL
‘Table Inhibition
of Tryptophan
Inhibitors
RESEARCH COMMUNICATIONS
II.
Pyrrolase
Activity
By Chelating
Agents
Concentrations
Inhibition
8 -Hydroxyquinoline
1 x 10-2
0
d , d’-dipyridyl
1 x 10-Z
0
E,DTA
1 x 10 -2
11
1 x 10 -3
11
1 x 10-s 1 x 10-4
100
Sodium
Azide
E:CN
(%)
80
1)iethyldithiocarbamate
1 x 10 -2 1 x 10 -3
68 12
S alicylaldoxime
8 4
x 1O-3 x 10-8 1 x 10-3
86 62 27
(Zuprizone
4
x 1o-3
52
13athocuproinedisulfonate
9 x 10-4 3 x 10-4 9 x 10-5
100 39 22
The enzyme was preincubated with chelating agents for 10 minutes ;tnd then L-tryptophan, hematin and ascorbate +ere added. The rate of ::ormylkynurenine formation was then measured spectrophotometrically as previously described (Fei.gelson et Cuprizone, being water -- al., 1965). :.nsoluble, was dissolved into 50% ethanol; in this control experiment, the same volume of 50% ethanol was added to the incubation mixture.
that
the substrates
are
bound
enzyme. between, pyrrolase
of tryptophan
pyrrolase,
oxygen
and tryptophan,
respectively
to the cuprous
and hematin
The
catalytic
of, and the possible
the are
specific enzyme
bound
under
further
copper
roles
and hematin
investigation.
301
components
moities
of the
interaction in tryptophan
Vol. 21, No. 4, 1965
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES Reagents Cuproine, Neocuproine, Diehl, H. and Smith G. F., “The Copper Bathocuproine” G. F. Smith Co., Columbus, Ohio (1958). Feigelson, P., Ishimura, Y., and Hayaishi, O., Biochem. Biophys. Res. Commun. 14:96 (1964). Feigelson, P., Ishimura Y., and Hayaishi, 0.) Biochim. Biophys. Acta, 95 283 (1965). P., J. Biol. Chem., 237, 1903 (1962). Greengard, 0.) and Feigelson, Congress of Biochemxtry (New York) Hayaishi, O., Sixth International -1.u. B. vol. 33, p. 31 (1964). McFarlane, W., Biochem. J., 24, 1022 (1932). edited by 0. Hayaishi, Academic Press Okunuki, K., in “Oxygenases” New York and London, p/409 (1962). Peterson, R., and Bollier, M., Anal. Chem., 27; 1195 (1955). Poillon, N. W., and Dawson, C.H., Biochim. Bgphys. Acta, 72, 27 (1963). Simonsen, S. H., and Burnett, H. M., Anal. Chem., 22; 1336 (1955). W. E., J. Biol. Chem., 234, 1162 (1959). Tanaka, T., and Knox, Biophys. Acta, 81, 201 (1964). Tokuyama, K., and Knox, W. E., Biochim.
302