Vol. 66, No. 3,1975
BIOCHEMICAL
A “REVERSE
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BURST” ACTIVE SITE FOR HUMAN CARBONIC William
M.
Mendez,
Jr.
TITRATION PROCEDURE ANHYDRASE B and E. T.
Departments of Chemistry University of Chicago Chicago, Illinois 60637 Received
August
Summary:
binds
constant
pH 7 and pH 10.5,
very
on the order
the enzyme.
strongly
Because
this
measuring
the rapid
hydrolysis
The enzyme Titration
due to the release
number ester
(HCAB) Materials
solutions,
and convenient
enzyme
In the present p-sulfamyl
by
under
readily
titrant,
by
catalyzed
by
have
been
to report
in a complementary of human
carbonic
“reverse
burst”
we term
the titration
of
1
enzymology. %urstsn
the stoichiometric
we wish
normality
for
of absorbance
sites
article
which
of modern
from
active
benzoate site
a method
site
PNP-SAB
procedures
objective
products
of the active
as an active
unbound
of chromogenic
2
but is not hydrolyzed
burst”).
a major
with
B (dissociation
stoichiometric
be used
the measurement
p-nitrophenyl
determination
may
benzoate
anhydrase
at pH 7.5),
involving
substrates of cases.
carbonic
of excess
remains
procedures
of suitable
ester
of rapid
sites
p-sulfamyl
is essentially
(“reverse
development
active
to human
the binding
conditions,
nucleophile
p-nitrophenyl
of 10 -9 M or less
accessible
an added
and Biochemistry
5,1975
Between
(PNP-SAB)
Kaisers
reactions
described
in a
the use of the approach anhydrase
in the B
titration.
and Methods Carbonic
anhydrase
was
Copyright D I9 75 b.v Academic Press. Inc. All rights of reproduction in any form reserved.
prepared
from 949
human
erythrocytes
by affinity
Vol. 66, No. 3, 1975
3
chromatography. method the
BIOCHEMICAL
The
of Armstrong correct
B isozyme
acid
all
-’
solvents
sulfamyl
bensoic
was ).
Found:
&&. C,
for
the
tion
of
and
in
After
from
ethanol.
for
C13H10N206S: H,
solutions
S,
also
exchange exhibited of
ethanol
ester
Ewith
ester
with
melted
48.46;
and
p-Nitrophenyl
the
H,
of
25’. from
use.
2-hydroxy
constant
of p-nitrophenol
N,
excess
H20,
169-170°
at
3.1;
E-
8. 69;
(un-
S,
9.95.
9. 90. was
a pK
was
to
pure C
8. 74;
and
7. 7,
precipitating The
ion
rate
recrystallized
coupling
of 2..nitrophenol species
enzyme
DCC
order
pH
prior
acid
concentration
phenolate
dry-distilled
the
hydrolysis
a second
was
by
the
buffer,
(Eastman)
3.09;
by
homogeneous,
with
T ris-sulfate
DMF.
purified
catalyzed
sultone5
prepared
H,
was
RESEARCH COMMUNICATIONS
electrophoretically
was
Calcd 48.57;
The
M
were
recrystallized
corr.
for
calculated
using
E-nitrophenol
measured
of
7.15.
E 4oo 6
= 18,
The
100
concentra-
spectrophotometrically,
e280
7
48,900. Results
and We
Discussion
have
is
bound
tightly
if
at all,
by
sulfate
discovered in the
‘The
until
approximately
at which to
pH
resulted
lease.
the
over
7.4,
the no
formation
the
an
fashion
to
range
7-10.
1 x 10
the
overall
amounts
of
amounts
buffer-catalyzed
p-sulfamyl
of in
of increased
of
pH
addition
increase
equimolar the
E-nitrophenyl
a stoichiometric
in
addition
point
that
enzyme
buffer,
PNP-SAB
due
0.1
substrate
sulfamylbenaoate
it
in
sultone
organic
was
acid
M-‘set
The
which
composition,
5-nitro-a-toluenesulfonic 7. 6 x lo5
4
et al.
amino
AND BIOPHYSICAL
of
hydrolytically
but
5.
is
For
rate
example,
slowly, in
Tris-
-5
of p-nitrophenol
enzyme
was and
of
PNP-SAB,
hydrolyzed
MHCABto3.5xlO
enzyme
stable
950
HCAB
-6
hydrolysis
benzoate,
the complex
without
ester ester
were ceased,
between
M re-
effect present, evidently PNP-SAB
=
Vol. 66, No. 3, 1975
and
the
enzyme.
catalyze cess
BIOCHEMICAL
Over
the
hydrolysis
PNP-SAB,
it
the
is
possible
from
enzyme’s
active
site
centration
of the
unbound
initial
of the
(tot
HCAB In
of
centration of
slower,
to
ester
containing
a ‘burst’! the
nucleophiles
due
substrate By
to
simple in
is
and
rapid
release
-not
bound
ex-
to
of
manner
from
the
active
site
normality
increase
in
the
the
the
subtraction this
substrate,
HCAB the
which
which
con-
established. it
was
found
sufficient was
initially
to
that
accompanied
by
at time early
p-nitrophenol
in
rapid
a significant,
though
release
Thus,
(t) the
was
the
a conveniently
ester.
unbound a point
an
cause
complexed
ester
t = 0 from
when
solutions
of the
ester
of
adding
determined
nucleophile
concentration
complexed
of
examined
of the
back
part
by
burst”).
be
cases
uncomplexed
extrapolate
obtain
substrate
can
hydrolysis the
that
to
concentration
of added
rolysis
mine
al)
the
7-10.5
to
(“reverse
solution
all
range
of PNP-SAB
of p-nitrophenolate
known
pH
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
= 0,
in
it
was
hydmuch to
deter-
necessary
hydrolysis very
order
con-
of
nearly
the
to
initially
zero-order,
:‘as
is
i-llustrated With
according
in the
to apparent
the
absence
in
0.1
M piperazine-N,
proved
PNP-SAB
to 1.
give
1.
nucleophiles
in
Table
Figure
of
tested first-order
enzyme.
Among N’
the
most
Under
these
was
complete
the
hydrolysis
of
kinetics
and
these
nucleophiles
-bis(Z-ethanesulfonic accurate
conditions, in
about
occurred
reproducible 25.
7 min,
0’ and
ester at the
10 mM acid)
and at
unbound
the the
results
), as
hydrolysis
of are
Whether the slow hydrolysis of the initially complexed reaction on the enzyme or corresponds to the slow dissociation PNP-SAB complex, followed by rapid scavenging by the added being investigated in our laboratory.
951
same
rate
as
hydroxylamine
(Pipes results
proceeded
pH
7.5,
illustrated
in
uncomplexed accurate
and
ester involves of the enzyme. nucleophile is
Vol. 66, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
NITROPHENOL RELEASED
B
TIME
Figure
1. --Schematic
representation
titration
of HCAB
unbound
ester
slower
enabling at time
[Unbound
of PNP-SAB ant inhibition
with
to measure HCAB
acid
to be on the order
of 10
not only
but also
appears
Thus,
site
the hydrolysis
of
A and B the much to approximate of unbound
[Enzyme
active
concentration
the dissociation
zeroester
sites]
to be
= [Total
Ester
failed low
-9
constant,
because
enzyme
“reverse this
compound
PNP-SAB
Because
burst”
for
the complex
of the concomit-
of 2-hydroxy-5-nitro-abound
concentrations. **
Kd,
the extent
hydrolysis
M or less.
to carry
to employ
of 10 PM.
at pH 7.5 by determining
sultone
at very
SAB
= 0.
1% at an enzyme
even
possible
points
the concentration
of the enzyme-catalyzed
toluenesulfonic metrically,
ester
A,
active
Ester).
to within
An attempt
burst”
At point
Between
of bound
kinetics,
determined
PNP-SAB.
is complete.
hydrolysis
order
reproducible
with
of a “reverse
The
almost value
Kd is very
active
as an inhibitor
site
titrations in rate
stoichioof Kd appears it is
small
with assay
PNPmeasure-
The inhibition of carbonic anhydrase action by several alkyl esters of p-sulfamyl benzoate has been measured and Kd values for the enzyme-inhibitor complexes as low as about lo-lo M have been measured. 8
952
Vol. 66, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
C c s
953
Vol. 66, No. 3, 1975
ments
which
can
centration,
as
sulfonic
acid
technique
BIOCHEMICAL
lead
to the
illustrated
is
far
is
of
even
Figure
2. --Active assay
in
with
hydrolysis
10
M
-6
assay
of
the
2 where
enzyme
the
presence
site
4. q- However,
the
have
achieved
by
found the
that “reverse
of a comparable
titration
of HCAB
the
con-
‘Yeverse
accurate
the
latter
burst”
concentration
with
pH
7.
was
68,
1% CH3CN,
O”.
a rate The
sultone. nm
The 25.
another
employing
at 410
spectrophotometer.
using of
PNP-SAB
followed
site
method
acid
substrate
stopped-flow
active
burst”
2-hydroxy-5-nitro-a-toluenesulfonic
Tris-sulfate,
site
2-hydroxy-5-nitro-a-toluene-
substrate.
we
be
of
active
convenient.
can
Aminco-Morrow 0.1
Figure
studies
HCAB
PNP-SAB
the
more
In preliminary titration
determination
in
s&one
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on an buffer
[Enzyme]
was = 4.11
M.
The use of an inhibitor in conjunction with a rate assay has conventionally applied in measurements of the active site normalities carbonic anhydrase solutions, as exemplified by Kernohan’ s titration bovine carbonic anhydrase solutions with ethoxazolamide. 9
954
been of of
x
Vol. 66, No. 3, 1975
BIOCHEMICAL
cr-chymotrypsin,
enzyme, continuing
our
isomers
which
investigation
with
other
AND BIOPHYSICAL
reacts
of the
carbonic
with
this
interaction
anhydrase
RESEARCH COMMUNICATIONS
ester.
of
We
PNP-SAB
are and
actively its
structural
species.
Acknowledgments W. National K.
M.
M.
was
Institute
T.
supported
of
Douglas
and
by
General Y.
Training
Medical
Nakagawa
Grant
Sciences. for
their
No. We
advice
GM-424 wish
and
to
from thank
the Drs.
encouragement.
References 1)
2)
F.
J. KLzdy and G. E. Perlman
M.
L. to
3)
W.
M.
4)
J.
5)
K.-W.
6)
J.
7)
K.-W.
8) 9)
Bender, Proteins,
E. T. Kaiser, and L. Lorand,
“Mechanisms ” Wiley-Interscience
Mendez,
Jr.
and
McD. Armstrong, J. Biol. Chem.,
T. J.
Lo B.
and
E.
Gibbons Lo,
D. 241, T.
and
Maren,
C.
Kernohan,
of
T.
T.
Physiol. Biochim.
Kaiser,
9
..
Chem Edsall,
Thesis,
-47,
pp.
J.
A.
of 595
Biophys.
in
J.
834 Chem.,
Chicago
96,
T.
Edsall,
3502
(1963).
(1966). 238,
(1968).
304
Protons
preparation.
(1967). Acta,
955
from
Verpoorte,
Commun.,
Vol. XIX, (1970), p.
Catalysis 397-455.
manuscript
Biol.
J
University Rev.,
in Enzymology, Academic Press
Homogeneous (1971),
V. Myers 5137 (1966
Kaiser, J.
Ph.D.
H.
E.
Methods eds.,
(1965).
3.
BIOCHEMICAL
A “REVERSE
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BURST” ACTIVE SITE FOR HUMAN CARBONIC William
M.
Mendez,
Jr.
TITRATION PROCEDURE ANHYDRASE B and E. T.
Departments of Chemistry University of Chicago Chicago, Illinois 60637 Received
August
Summary:
binds
constant
pH 7 and pH 10.5,
very
on the order
the enzyme.
strongly
Because
this
measuring
the rapid
hydrolysis
The enzyme Titration
due to the release
number ester
(HCAB) Materials
solutions,
and convenient
enzyme
In the present p-sulfamyl
by
under
readily
titrant,
by
catalyzed
by
have
been
to report
in a complementary of human
carbonic
“reverse
burst”
we term
the titration
of
1
enzymology. %urstsn
the stoichiometric
we wish
normality
for
of absorbance
sites
article
which
of modern
from
active
benzoate site
a method
site
PNP-SAB
procedures
objective
products
of the active
as an active
unbound
of chromogenic
2
but is not hydrolyzed
burst”).
a major
with
B (dissociation
stoichiometric
be used
the measurement
p-nitrophenyl
determination
may
benzoate
anhydrase
at pH 7.5),
involving
substrates of cases.
carbonic
of excess
remains
procedures
of suitable
ester
of rapid
sites
p-sulfamyl
is essentially
(“reverse
development
active
to human
the binding
conditions,
nucleophile
p-nitrophenyl
of 10 -9 M or less
accessible
an added
and Biochemistry
5,1975
Between
(PNP-SAB)
Kaisers
reactions
described
in a
the use of the approach anhydrase
in the B
titration.
and Methods Carbonic
anhydrase
was
Copyright D I9 75 b.v Academic Press. Inc. All rights of reproduction in any form reserved.
prepared
from 949
human
erythrocytes
by affinity
Vol. 66, No. 3, 1975
3
chromatography. method the
BIOCHEMICAL
The
of Armstrong correct
B isozyme
acid
all
-’
solvents
sulfamyl
bensoic
was ).
Found:
&&. C,
for
the
tion
of
and
in
After
from
ethanol.
for
C13H10N206S: H,
solutions
S,
also
exchange exhibited of
ethanol
ester
Ewith
ester
with
melted
48.46;
and
p-Nitrophenyl
the
H,
of
25’. from
use.
2-hydroxy
constant
of p-nitrophenol
N,
excess
H20,
169-170°
at
3.1;
E-
8. 69;
(un-
S,
9.95.
9. 90. was
a pK
was
to
pure C
8. 74;
and
7. 7,
precipitating The
ion
rate
recrystallized
coupling
of 2..nitrophenol species
enzyme
DCC
order
pH
prior
acid
concentration
phenolate
dry-distilled
the
hydrolysis
a second
was
by
the
buffer,
(Eastman)
3.09;
by
homogeneous,
with
T ris-sulfate
DMF.
purified
catalyzed
sultone5
prepared
H,
was
RESEARCH COMMUNICATIONS
electrophoretically
was
Calcd 48.57;
The
M
were
recrystallized
corr.
for
calculated
using
E-nitrophenol
measured
of
7.15.
E 4oo 6
= 18,
The
100
concentra-
spectrophotometrically,
e280
7
48,900. Results
and We
Discussion
have
is
bound
tightly
if
at all,
by
sulfate
discovered in the
‘The
until
approximately
at which to
pH
resulted
lease.
the
over
7.4,
the no
formation
the
an
fashion
to
range
7-10.
1 x 10
the
overall
amounts
of
amounts
buffer-catalyzed
p-sulfamyl
of in
of increased
of
pH
addition
increase
equimolar the
E-nitrophenyl
a stoichiometric
in
addition
point
that
enzyme
buffer,
PNP-SAB
due
0.1
substrate
sulfamylbenaoate
it
in
sultone
organic
was
acid
M-‘set
The
which
composition,
5-nitro-a-toluenesulfonic 7. 6 x lo5
4
et al.
amino
AND BIOPHYSICAL
of
hydrolytically
but
5.
is
For
rate
example,
slowly, in
Tris-
-5
of p-nitrophenol
enzyme
was and
of
PNP-SAB,
hydrolyzed
MHCABto3.5xlO
enzyme
stable
950
HCAB
-6
hydrolysis
benzoate,
the complex
without
ester ester
were ceased,
between
M re-
effect present, evidently PNP-SAB
=
Vol. 66, No. 3, 1975
and
the
enzyme.
catalyze cess
BIOCHEMICAL
Over
the
hydrolysis
PNP-SAB,
it
the
is
possible
from
enzyme’s
active
site
centration
of the
unbound
initial
of the
(tot
HCAB In
of
centration of
slower,
to
ester
containing
a ‘burst’! the
nucleophiles
due
substrate By
to
simple in
is
and
rapid
release
-not
bound
ex-
to
of
manner
from
the
active
site
normality
increase
in
the
the
the
subtraction this
substrate,
HCAB the
which
which
con-
established. it
was
found
sufficient was
initially
to
that
accompanied
by
at time early
p-nitrophenol
in
rapid
a significant,
though
release
Thus,
(t) the
was
the
a conveniently
ester.
unbound a point
an
cause
complexed
ester
t = 0 from
when
solutions
of the
ester
of
adding
determined
nucleophile
concentration
complexed
of
examined
of the
back
part
by
burst”).
be
cases
uncomplexed
extrapolate
obtain
substrate
can
hydrolysis the
that
to
concentration
of added
rolysis
mine
al)
the
7-10.5
to
(“reverse
solution
all
range
of PNP-SAB
of p-nitrophenolate
known
pH
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
= 0,
in
it
was
hydmuch to
deter-
necessary
hydrolysis very
order
con-
of
nearly
the
to
initially
zero-order,
:‘as
is
i-llustrated With
according
in the
to apparent
the
absence
in
0.1
M piperazine-N,
proved
PNP-SAB
to 1.
give
1.
nucleophiles
in
Table
Figure
of
tested first-order
enzyme.
Among N’
the
most
Under
these
was
complete
the
hydrolysis
of
kinetics
and
these
nucleophiles
-bis(Z-ethanesulfonic accurate
conditions, in
about
occurred
reproducible 25.
7 min,
0’ and
ester at the
10 mM acid)
and at
unbound
the the
results
), as
hydrolysis
of are
Whether the slow hydrolysis of the initially complexed reaction on the enzyme or corresponds to the slow dissociation PNP-SAB complex, followed by rapid scavenging by the added being investigated in our laboratory.
951
same
rate
as
hydroxylamine
(Pipes results
proceeded
pH
7.5,
illustrated
in
uncomplexed accurate
and
ester involves of the enzyme. nucleophile is
Vol. 66, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
NITROPHENOL RELEASED
B
TIME
Figure
1. --Schematic
representation
titration
of HCAB
unbound
ester
slower
enabling at time
[Unbound
of PNP-SAB ant inhibition
with
to measure HCAB
acid
to be on the order
of 10
not only
but also
appears
Thus,
site
the hydrolysis
of
A and B the much to approximate of unbound
[Enzyme
active
concentration
the dissociation
zeroester
sites]
to be
= [Total
Ester
failed low
-9
constant,
because
enzyme
“reverse this
compound
PNP-SAB
Because
burst”
for
the complex
of the concomit-
of 2-hydroxy-5-nitro-abound
concentrations. **
Kd,
the extent
hydrolysis
M or less.
to carry
to employ
of 10 PM.
at pH 7.5 by determining
sultone
at very
SAB
= 0.
1% at an enzyme
even
possible
points
the concentration
of the enzyme-catalyzed
toluenesulfonic metrically,
ester
A,
active
Ester).
to within
An attempt
burst”
At point
Between
of bound
kinetics,
determined
PNP-SAB.
is complete.
hydrolysis
order
reproducible
with
of a “reverse
The
almost value
Kd is very
active
as an inhibitor
site
titrations in rate
stoichioof Kd appears it is
small
with assay
PNPmeasure-
The inhibition of carbonic anhydrase action by several alkyl esters of p-sulfamyl benzoate has been measured and Kd values for the enzyme-inhibitor complexes as low as about lo-lo M have been measured. 8
952
Vol. 66, No. 3, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
C c s
953
Vol. 66, No. 3, 1975
ments
which
can
centration,
as
sulfonic
acid
technique
BIOCHEMICAL
lead
to the
illustrated
is
far
is
of
even
Figure
2. --Active assay
in
with
hydrolysis
10
M
-6
assay
of
the
2 where
enzyme
the
presence
site
4. q- However,
the
have
achieved
by
found the
that “reverse
of a comparable
titration
of HCAB
the
con-
‘Yeverse
accurate
the
latter
burst”
concentration
with
pH
7.
was
68,
1% CH3CN,
O”.
a rate The
sultone. nm
The 25.
another
employing
at 410
spectrophotometer.
using of
PNP-SAB
followed
site
method
acid
substrate
stopped-flow
active
burst”
2-hydroxy-5-nitro-a-toluenesulfonic
Tris-sulfate,
site
2-hydroxy-5-nitro-a-toluene-
substrate.
we
be
of
active
convenient.
can
Aminco-Morrow 0.1
Figure
studies
HCAB
PNP-SAB
the
more
In preliminary titration
determination
in
s&one
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on an buffer
[Enzyme]
was = 4.11
M.
The use of an inhibitor in conjunction with a rate assay has conventionally applied in measurements of the active site normalities carbonic anhydrase solutions, as exemplified by Kernohan’ s titration bovine carbonic anhydrase solutions with ethoxazolamide. 9
954
been of of
x
Vol. 66, No. 3, 1975
BIOCHEMICAL
cr-chymotrypsin,
enzyme, continuing
our
isomers
which
investigation
with
other
AND BIOPHYSICAL
reacts
of the
carbonic
with
this
interaction
anhydrase
RESEARCH COMMUNICATIONS
ester.
of
We
PNP-SAB
are and
actively its
structural
species.
Acknowledgments W. National K.
M.
M.
was
Institute
T.
supported
of
Douglas
and
by
General Y.
Training
Medical
Nakagawa
Grant
Sciences. for
their
No. We
advice
GM-424 wish
and
to
from thank
the Drs.
encouragement.
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