Vol.68,No.3,1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ASCORBATE-2-PHOSPHATE INHIBITION OF ASCORBATE-P-SULFATE SULFOHYDROLASE FROM BOVINE LIVER Russell W. Carlson, Mancourt Downing, Paul A. Seib, and Bert M. Tolbert Department of Chemistry University of Colorado Boulder, Colorado 80309 and Department of Grain Science and Industry Kansas State University Manhattan, Kansas 66506
Received
December
8,1975
Swmary . The hydrolysis of ascorbate-2-sulfate by the enzyme, ascorbate-2-sulfate sulfohydrolase, purified from bovine liver has been shown to be powerfully inhibited by ascorbate-2-phosphate. The inhibition by ascorbate phosphate is competitive with a K of 0.3 vM. Na2HP0, also inhibits by an apparent non-competitive proces 5 . The NazHP04 concentration at 50% inhibition is 7.7 FM. A possible control role for ascorbate phosphate in ascorbate biochemistry is suggested.
Ascorbate-P-sulfate which
was first
found
man, monkeys, storage
form
rats,
a crude
sulfate
ized
from
bovine
both
of these
fied
with
cases,
hydrolase
Copyright AlZ rights
the
arylsulfatase
are
Both
is
A preparation
from
(unpublished
sulfate
to a lesser
bovine
liver
In the
then,
and character(9,
10,
for
In
11).
activity
co-purisulfate
purified
arylsul-
and arylsulfatase
and by NazHP04. ascorbate
case of the marine 949
(2).
by Bullen Since
p-nitrocatechol
than
of
in trout
(6).
gastropod
using
acid
a
observed
purified
sulfohydrolase
degree
is probably
was first
sufohydrolase
phosphate
in the urine
scurvy
is a good substrate
by ascorbate
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
relieves
a marine
measured
sulfate
data).
It
has been partially
sulfate
of ascorbic
then
2, 3, 4, 5).
sulfate
ascorbate
inhibited
and since
of ascorbate
ascorbate
metabolite
and in fact
A activity
inhibited
(1,
8) and from
Ascorbate
A (12).
arylsulfatase
(7,
cysts
pigs
sulfohydrolase liver
occurring
shrimp
in trout,
hydrolysis
as a substrate.
activities
and guinea
arylsulfatase
ascorbate
fatase
in brine
of ascorbate
The enzymic using
is a naturally
However,
sulfate
gastropod,
sulfoascorbate
Vol.68No.3,
1976
sulfate
sulfohydrolase
several
nucleotides We report
sulfate
is
is
inhibited
strongly
a very
sulfohydrolase
Na2HPOr.
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
by inorganic
phosphate
and by
purified
ascorbate
(11).
here
The inhibition
BIOCHEMICAL
powerful
from
bovine
by ascorbate The possible
inhibition liver
phosphate
role
of a highly
by ascorbate is
stronger
of ascorbate
phosphate
than
phosphate
the
and Na2HP04.
inhibition
in ascorbate
by
biochemistry
discussed. MATERIALS
Ascorbate-Z-sulfate ascorbate-2-phosphate using (CH3)3N*S03
AND METHODS
and Aseorbate-2-phosphate. are prepared by direct and POC13 respectively.
(5)
sulfation
Ascorbate-P-sulfate or phosphorylation
and
Ascorbate-Z-sulfate
SuZfohydrwZase. The enzyme used in these studies has been purified 40,000 fold from bovine liver by orocedures which are in preparation for publication. Arylsulfatase A activity co-purifies with ascorbate sulfate sulfohydrolase throughout these procedures. Enzyme Assay. The enzyme assay consists of following the reduction of 2,6-dichloroindophenol by the ascorbate which is produced during the enzymic hydrolysis. The decrease in absorbance is followed at 516 nm which is the isobestic point of 2,6-dichloroindophenol. The pH of the assay reaction is pH 4.8 which is also the optimum. Since ascorbate sulfate is slowly acid hydrolyzed at this pH, a non-enzymic hydrolysis is used as a control. In the kinetic studies, 1.5 ml of 0.15 mM 2,6-dichloroindophenol in 0.05 M KAc pH 4.8 are placed in a 3 ml cuvette. To this is added an appropriate amount of 0.5 M NaAc or KAc, and water to give a 70 mM dipotassium ascorbate sulfate, total volume of 2 ml and an ionic strength of 0.1. Trisodium ascorbate phosphate and Na2HP04 are added so that their final concentrations are from 0.2 to 0.4 uM and from 7.7 to 14.8 uM respectively. To the cuvette is added 0.05 ml of a 40/l dilution of purified enzyme solution which had an initial protein concentration of 0.26 mg/ml (determined by the Lowry method using BSA as a standard). For each substrate concentration the non-enzymic hydrolysis rate is subtracted from the hydrolysis rate with the enzyme to give the true rate of enzymic hydrolysis. The rate of reaction at each substrate concentration is measured three to four times. RESULTS AND DISCUSSION Figure the
inhibition
KI of 0.3 tion
point
bition. systematic tion
1 (Top)
shows
that
by ascorbate uM.
Figure
is
to the
However,
the
error
could
by Na2HP04 is
the
phosphate
1 (Bottom) left area
Km for
of the
ascorbate
is
competitive
shows the l/v
axis
which
competitive
inhibition.
questionable.
The reaction
Na2HP04.
950
close
powerful
to the
inhibited
with
zero
line type
a
The intersec-
non-competitive
Thus the is
12 mM, and that
by NanHPOs.
indicates
is
is
and very
inhibition
of intersection
give
sulfate
inhiand a small
of inhibi-
50% by 7.7 PM
Vol.68,No.3,1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 1. Top: A Lineweaver-Burke plot showing the inhibition of ascorbate2-sulfate sulfohydrolase by ascorbate-2-phosphate. Bottom: A LineweaverBurke plot showing the inhibition of ascorbate-2-sulfate sulfohydrolase by Na,HPO,. The dashed lines are-with the indicated inhibitor concentration and the solid lines are without inhibitor.
These
results
are
phosphate
is approximately
Na2HP0,+.
The KI for
The powerful phosphate tightly than
is
for
is different quite
a KI of 0.3
phosphate
Na2HP04 and that from
Na,HP04
sulfate
that the
suggests
The inhibition than
is 40,000
FM shows that
The fact
I.
more powerful
of ascorbate
to the enzyme. it
on Table
25 fold
ascorbate
inhibition with
summarized
type that
the
fold
lower
is
binding
than
is
25 fold
of inhibition
by the
Km.
by ascorbate
phosphate
binding
the
inhibition
sulfohydrolase
ascorbate
this
by ascorbate
bound
very
more powerful
by ascorbate of ascorbate
phosphate phosphate
specific. Ascorbate
phosphate
in neutral
aqueous
pig
It
(13).
is
solution. subject
is an interesting It to rapid
molecule.
can be used to cure hydrolysis
951
by alkaline
It
is
scurvy
stable
for
weeks
in the guinea
phosphatase
(unpub-
is
Vol.68,No.3,1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE I Inhibition 2-Phosphate.
of Ascorbate-2-Sulfate
None
KI = 0.3
NazHPO,,
lished
inhibition
data)
yet
properties,
coupled
reported
above,
an active
form
sulfatase
be present
sulfate.
(2)
if
the
of the are
published
that
under
Ascorbate in which
similar
paper,
Details
A enzymes
These
phosphate may function
compound
sulfate is
as
for
sulfate (EC 3.1.6.1)
the
scurvy from
in trout
ascorbate
control
of the
in this
paper.
sulfohydrolase, and its of this
ascorbate
of such a control
derived
described
of the properties
for
prevents
candidate
ascorbate
tissue.
phosphate
An example
to that
energy
conditions.
ascorbate
enzyme is
in
high
of ascorbate
as a control report.
in the
is unstable
in biological
biological
in this
it
explained
be a moderately
ascorbate
is a likely
arylsulfatase
time
is
since
binding
phosphate
in this
uncertain.
should
tight
serve
trout.
by a process
enzyme described be part
could
in the
Ascorbate
regeneration
acid
This
phosphatases
life
us to suggest
described
Non-competitivea
questionable.
phosphate
the very
phosphate
function
and salmon
roles
with
lead
is
have a short
of ascorbic
Ascorbate
might
would
Competitive
many other
Ascorbate
it
UM
by NazHPO,
and probably fluids.
compound,
---
= 7.7 PM
[I'50%
of
biological
Type of Inhibition
Km = 12 mM
Ascorbate-2-phosphate
type
by NazHPOI, and by Ascorbate-
KI or Inhibitor Concentration at 50% Inhibition [I],,,
Inhi bitor
aThe text.
Sulfohydrolase
major
enzyme will
The
seems to biological be
later. ACKNOWLEDGMENT
This Office
work
of the
is supported
in part
by a contract
U. S. Army.
952
with
the
Surgeon
General's
Vol.68,No.3,1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8.
1;: 11. 12. 13.
Mead, C. G., and Finamore, F. S. (1969) Biochem. 8, 2652-2655. Halver, J. E., Smith, R. R., Tolbert, 8. M., and Baker, E. M. (1975) Ann. N. Y. Acad. Sci. 258, 81-102. Baker, E. M., Hammer, D. C., March, S. C., Tolbert, B. M., and Canham, J. E. (1971) Science, 173, 826-827. March, S. C. (1972) "Some Aspects of the Synthesis, Analysis and Occurrence of Ascorbate-2-Sulfate and its Metabolism in Rats," Ph. 0. Thesis, University of Colorado. Tolbert, B. M., Downing, M., Carlson, R. W., Knight, M. K., and Baker, E. M. (1975) Ann. N. Y. Acad. Sci 258, 48-69. Bullen, W. (1972) "Ascorbate-2-Sulfate Sulfohydrolase," Master's Thesis, University of Colorado. Carlson, R. W. (1974) "Ascorbate-Z-Sulfate Sulfohydrolase: Partial Purification and Characterization," Master's Thesis, University of Colorado. Carlson, R. W., Downing, M., and Tolbert, 8. M. (1974) Fed. Proc. 33, 1377. Hatanaka, H., Ogawa, Y., and Egami, F. (1974) J. Biochem. 75, 861-866. Hatanaka, H., Ogawa, Y., and Egami, F. (1975) J. Biochem. 77, 353-359. Hatanaka, H., Ogawa, Y., and Egami, F. (1975) J. Biochem. 77, 807-810. Roy, A. B. (1975) Biochim. Biophys. Acta. 377, 356-363. Cutolo, E., and Larizza, A. (1961) Gass. Chim. Ital. 91, 964-972.
953
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ASCORBATE-2-PHOSPHATE INHIBITION OF ASCORBATE-P-SULFATE SULFOHYDROLASE FROM BOVINE LIVER Russell W. Carlson, Mancourt Downing, Paul A. Seib, and Bert M. Tolbert Department of Chemistry University of Colorado Boulder, Colorado 80309 and Department of Grain Science and Industry Kansas State University Manhattan, Kansas 66506
Received
December
8,1975
Swmary . The hydrolysis of ascorbate-2-sulfate by the enzyme, ascorbate-2-sulfate sulfohydrolase, purified from bovine liver has been shown to be powerfully inhibited by ascorbate-2-phosphate. The inhibition by ascorbate phosphate is competitive with a K of 0.3 vM. Na2HP0, also inhibits by an apparent non-competitive proces 5 . The NazHP04 concentration at 50% inhibition is 7.7 FM. A possible control role for ascorbate phosphate in ascorbate biochemistry is suggested.
Ascorbate-P-sulfate which
was first
found
man, monkeys, storage
form
rats,
a crude
sulfate
ized
from
bovine
both
of these
fied
with
cases,
hydrolase
Copyright AlZ rights
the
arylsulfatase
are
Both
is
A preparation
from
(unpublished
sulfate
to a lesser
bovine
liver
In the
then,
and character(9,
10,
for
In
11).
activity
co-purisulfate
purified
arylsul-
and arylsulfatase
and by NazHP04. ascorbate
case of the marine 949
(2).
by Bullen Since
p-nitrocatechol
than
of
in trout
(6).
gastropod
using
acid
a
observed
purified
sulfohydrolase
degree
is probably
was first
sufohydrolase
phosphate
in the urine
scurvy
is a good substrate
by ascorbate
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
relieves
a marine
measured
sulfate
data).
It
has been partially
sulfate
of ascorbic
then
2, 3, 4, 5).
sulfate
ascorbate
inhibited
and since
of ascorbate
ascorbate
metabolite
and in fact
A activity
inhibited
(1,
8) and from
Ascorbate
A (12).
arylsulfatase
(7,
cysts
pigs
sulfohydrolase liver
occurring
shrimp
in trout,
hydrolysis
as a substrate.
activities
and guinea
arylsulfatase
ascorbate
fatase
in brine
of ascorbate
The enzymic using
is a naturally
However,
sulfate
gastropod,
sulfoascorbate
Vol.68No.3,
1976
sulfate
sulfohydrolase
several
nucleotides We report
sulfate
is
is
inhibited
strongly
a very
sulfohydrolase
Na2HPOr.
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
by inorganic
phosphate
and by
purified
ascorbate
(11).
here
The inhibition
BIOCHEMICAL
powerful
from
bovine
by ascorbate The possible
inhibition liver
phosphate
role
of a highly
by ascorbate is
stronger
of ascorbate
phosphate
than
phosphate
the
and Na2HP04.
inhibition
in ascorbate
by
biochemistry
discussed. MATERIALS
Ascorbate-Z-sulfate ascorbate-2-phosphate using (CH3)3N*S03
AND METHODS
and Aseorbate-2-phosphate. are prepared by direct and POC13 respectively.
(5)
sulfation
Ascorbate-P-sulfate or phosphorylation
and
Ascorbate-Z-sulfate
SuZfohydrwZase. The enzyme used in these studies has been purified 40,000 fold from bovine liver by orocedures which are in preparation for publication. Arylsulfatase A activity co-purifies with ascorbate sulfate sulfohydrolase throughout these procedures. Enzyme Assay. The enzyme assay consists of following the reduction of 2,6-dichloroindophenol by the ascorbate which is produced during the enzymic hydrolysis. The decrease in absorbance is followed at 516 nm which is the isobestic point of 2,6-dichloroindophenol. The pH of the assay reaction is pH 4.8 which is also the optimum. Since ascorbate sulfate is slowly acid hydrolyzed at this pH, a non-enzymic hydrolysis is used as a control. In the kinetic studies, 1.5 ml of 0.15 mM 2,6-dichloroindophenol in 0.05 M KAc pH 4.8 are placed in a 3 ml cuvette. To this is added an appropriate amount of 0.5 M NaAc or KAc, and water to give a 70 mM dipotassium ascorbate sulfate, total volume of 2 ml and an ionic strength of 0.1. Trisodium ascorbate phosphate and Na2HP04 are added so that their final concentrations are from 0.2 to 0.4 uM and from 7.7 to 14.8 uM respectively. To the cuvette is added 0.05 ml of a 40/l dilution of purified enzyme solution which had an initial protein concentration of 0.26 mg/ml (determined by the Lowry method using BSA as a standard). For each substrate concentration the non-enzymic hydrolysis rate is subtracted from the hydrolysis rate with the enzyme to give the true rate of enzymic hydrolysis. The rate of reaction at each substrate concentration is measured three to four times. RESULTS AND DISCUSSION Figure the
inhibition
KI of 0.3 tion
point
bition. systematic tion
1 (Top)
shows
that
by ascorbate uM.
Figure
is
to the
However,
the
error
could
by Na2HP04 is
the
phosphate
1 (Bottom) left area
Km for
of the
ascorbate
is
competitive
shows the l/v
axis
which
competitive
inhibition.
questionable.
The reaction
Na2HP04.
950
close
powerful
to the
inhibited
with
zero
line type
a
The intersec-
non-competitive
Thus the is
12 mM, and that
by NanHPOs.
indicates
is
is
and very
inhibition
of intersection
give
sulfate
inhiand a small
of inhibi-
50% by 7.7 PM
Vol.68,No.3,1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 1. Top: A Lineweaver-Burke plot showing the inhibition of ascorbate2-sulfate sulfohydrolase by ascorbate-2-phosphate. Bottom: A LineweaverBurke plot showing the inhibition of ascorbate-2-sulfate sulfohydrolase by Na,HPO,. The dashed lines are-with the indicated inhibitor concentration and the solid lines are without inhibitor.
These
results
are
phosphate
is approximately
Na2HP0,+.
The KI for
The powerful phosphate tightly than
is
for
is different quite
a KI of 0.3
phosphate
Na2HP04 and that from
Na,HP04
sulfate
that the
suggests
The inhibition than
is 40,000
FM shows that
The fact
I.
more powerful
of ascorbate
to the enzyme. it
on Table
25 fold
ascorbate
inhibition with
summarized
type that
the
fold
lower
is
binding
than
is
25 fold
of inhibition
by the
Km.
by ascorbate
phosphate
binding
the
inhibition
sulfohydrolase
ascorbate
this
by ascorbate
bound
very
more powerful
by ascorbate of ascorbate
phosphate phosphate
specific. Ascorbate
phosphate
in neutral
aqueous
pig
It
(13).
is
solution. subject
is an interesting It to rapid
molecule.
can be used to cure hydrolysis
951
by alkaline
It
is
scurvy
stable
for
weeks
in the guinea
phosphatase
(unpub-
is
Vol.68,No.3,1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE I Inhibition 2-Phosphate.
of Ascorbate-2-Sulfate
None
KI = 0.3
NazHPO,,
lished
inhibition
data)
yet
properties,
coupled
reported
above,
an active
form
sulfatase
be present
sulfate.
(2)
if
the
of the are
published
that
under
Ascorbate in which
similar
paper,
Details
A enzymes
These
phosphate may function
compound
sulfate is
as
for
sulfate (EC 3.1.6.1)
the
scurvy from
in trout
ascorbate
control
of the
in this
paper.
sulfohydrolase, and its of this
ascorbate
of such a control
derived
described
of the properties
for
prevents
candidate
ascorbate
tissue.
phosphate
An example
to that
energy
conditions.
ascorbate
enzyme is
in
high
of ascorbate
as a control report.
in the
is unstable
in biological
biological
in this
it
explained
be a moderately
ascorbate
is a likely
arylsulfatase
time
is
since
binding
phosphate
in this
uncertain.
should
tight
serve
trout.
by a process
enzyme described be part
could
in the
Ascorbate
regeneration
acid
This
phosphatases
life
us to suggest
described
Non-competitivea
questionable.
phosphate
the very
phosphate
function
and salmon
roles
with
lead
is
have a short
of ascorbic
Ascorbate
might
would
Competitive
many other
Ascorbate
it
UM
by NazHPO,
and probably fluids.
compound,
---
= 7.7 PM
[I'50%
of
biological
Type of Inhibition
Km = 12 mM
Ascorbate-2-phosphate
type
by NazHPOI, and by Ascorbate-
KI or Inhibitor Concentration at 50% Inhibition [I],,,
Inhi bitor
aThe text.
Sulfohydrolase
major
enzyme will
The
seems to biological be
later. ACKNOWLEDGMENT
This Office
work
of the
is supported
in part
by a contract
U. S. Army.
952
with
the
Surgeon
General's
Vol.68,No.3,1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8.
1;: 11. 12. 13.
Mead, C. G., and Finamore, F. S. (1969) Biochem. 8, 2652-2655. Halver, J. E., Smith, R. R., Tolbert, 8. M., and Baker, E. M. (1975) Ann. N. Y. Acad. Sci. 258, 81-102. Baker, E. M., Hammer, D. C., March, S. C., Tolbert, B. M., and Canham, J. E. (1971) Science, 173, 826-827. March, S. C. (1972) "Some Aspects of the Synthesis, Analysis and Occurrence of Ascorbate-2-Sulfate and its Metabolism in Rats," Ph. 0. Thesis, University of Colorado. Tolbert, B. M., Downing, M., Carlson, R. W., Knight, M. K., and Baker, E. M. (1975) Ann. N. Y. Acad. Sci 258, 48-69. Bullen, W. (1972) "Ascorbate-2-Sulfate Sulfohydrolase," Master's Thesis, University of Colorado. Carlson, R. W. (1974) "Ascorbate-Z-Sulfate Sulfohydrolase: Partial Purification and Characterization," Master's Thesis, University of Colorado. Carlson, R. W., Downing, M., and Tolbert, 8. M. (1974) Fed. Proc. 33, 1377. Hatanaka, H., Ogawa, Y., and Egami, F. (1974) J. Biochem. 75, 861-866. Hatanaka, H., Ogawa, Y., and Egami, F. (1975) J. Biochem. 77, 353-359. Hatanaka, H., Ogawa, Y., and Egami, F. (1975) J. Biochem. 77, 807-810. Roy, A. B. (1975) Biochim. Biophys. Acta. 377, 356-363. Cutolo, E., and Larizza, A. (1961) Gass. Chim. Ital. 91, 964-972.
953
