Vol. 83, No. 3, 1978
8lOCHEMlCAl
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
August 14,1978
Pages
ORGANOPHOSPHORUS
COMPOUNDS INHIBITORS
Padma
Received
June
AS ACTIVE
1178-1182
SITE-DIRECTED
OF ELASTASE
L. Nayak and Myron L. Division of Biochemistry Department of Chemistry Northwestern University Evanston, Illinois 60201
Bender
12,1978
SUMMARY Four ethyl R-nitrophenyl alkylphosphonates were studied for the inhibition of elastase. A pH-dependence study using the assay substrate BOC-Ala-ONp or the phosphonate inhibitors showed the participation of an ionizing group with an apparent pKa of 6.9 and a decrease of reaction or inhibition at higher Out of the four compounds investigated ethyl R-nitrophenyl pentylphosPH. phonate was found to be the best inhibitor of elastase as judged from the value which is the measure of inhibitory capacity, is the of k,/ KI. This value, highest reported so far for the inhibition of elastase. INTRODUCTION Elastase
is a pancreatic
(l),
remarkable
similarities
in its
to two
pancreatic
proteolytic
other
three
enzymes
nizes
large
of lysine alanine plete
differ
only
hydrophobic
remarkable almost
structures
degree identical,
containing
and the
process,
similar
geometrical
characterized
of similarity all
catalytic
groups
side
arrangements.
by the presence
sites.
The
binding
trypsin
binding
pocket
pocket
chains
is similar
0006-291X/78/0833-1178$01.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
to that
1178
recog-
side
residues
the com-
(3-6
).
There
is a They
catalytically
active
sites.
site
which
is acylated
serine,
pockets
and aspartic these adjacent
is hydrophobic of chymotrypsin
chains
especially
determined
enzymes
Furthermore,
of chymotrypsin
These
chymotrypsin
have
of histidine
of binding
and chymotrypsin.
aliphatic
of all three
an active
properties
positively-charged
of workers
in their
possesses
and biological
specificity:
trypsin small
which
trypsin
substrate
chains;
Several
three-dimensional
physical,
enzymes,
and elastase
derivatives.
(2) enzyme
chemical,
in their side
and arginine;
proteolytic
three
acid
are in the
in very
enzymes
are
to the catalytic and open,
the
but contains
a
BIOCHEMICAL
Vol. 83, No. 3, 1978
negatively
charged
elastase idue
aspartate
is narrow
near
A number
of investigations processes
emphysema
(8).
Inhibitors
of functional
elastase.
of primary could
provide
a means
in reality, make upon
use
they
been
shown
have
of studying
of the
both
in vivo.
center
Additionally,
and,
specificity
of valine
res-
be involved
therefore,
inhibitors
as tools their
with
in the attack
a
with
in the investideriva-
inhibitors
in a unique
in reacting
involved
with
radioactive
stoichiometric
mechanism
of purified
stoichiometrically
since
in
in pulmonary
Synthetic
served
these
factors
damage
to react
of the active
catalytic
may
for the characterization
have
the
pocket
of a bulky
elastase
tissue
compounds
substrates
same
that
elastic
role
as tags.
are
with
their
structure
be used
suggested
deducing
Organophosphorus
gation tives
have
tool
groups
and the binding
of the presence
a valuable
as for
RESEARCH COMMUNICATIONS
(7).
associated
provide
as well
variety
because
of the cavity
pathological
enzymes
ion in the bottom,
and shallower
the front
AND BIOPHYSICAL
way
can
because,
the enzyme, of the enzyme
substrates. In this
elastase butyl,
communication, with
n-pentyl
four
ethyl
we wish R-nitrophenyl
and n-hexyl),
to report
an account
alkylphosphonates
the phosphorus
analogs
of the inhibition (alkyl
of specific
= q-propyl, substrates
of nof
elastase.
MATERIALS
AND
METHODS
Porcine elastase was a Worthington Biochemical Corp. purified product used without further purification; its substrate BOC-Ala-ONp, was purchased Synthesis of ethyl R-nitrophenyl butylphosphonfrom Vega-Fox Biochemicals. ate (ENBP): Diethyl butyl-phosphonate was prepared by the application of Michaelis-Arbuzov reaction (9) in which equimolar quantities of triethylphosphite and n-butyl bromide were heated under reflux at 1500 to give diethyl butylphosphonate and ethyl bromide. Ethyl butylphosphonochloridate was synthesized by the reaction of diethyl butylphosphonate and phosphorus pentaEthyl R-nitrophenyl butylphosphonate was prepared by chloride in the cold. reacting ethyl butylphosphonochloridate with sodium R-~trophenoxide in toluene according to the method of Fukuto and Metcalf (10). The other three inhibitors which involve a change in the alkyl groups to l-propyl, e-pentyl and n-hexyl were synthesized according to the above procedure. Stock solutions of substrate (Spectra grade, Eastman Kodak
and inhibitor were prepared in acetonitrile Co. ). Buffer solutions were made from
1179
Vol. 83, No. 3, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.25 0.20 0.15 0.1
3
0.05
2 I 5
0
6
7
8
9
4
IO
0
5
x d4 M-’
2
PH
1
pH dependence of the inhibition of elastase by ENBP, 250, E = 2 x 10-6M; I = 1 x 10-4M.;, substrate = BOC-Ala-ONp = 5 x 10s5M. Determination of _t%e inhibition constant _o,f elastase by ENBP at 250, M, I = 0.25 - 2 x 10 M, substrate = BOC-Ala-ONp= pH 7. E = 2 x 10 5 x 1O-5 M.
1.
2.
reagent
grade
chemicals
of ionic
strength,
I - 0.1.
Elastase inhibition. A 25 to 100 fold molar excess of inhibitor over enzyme was used for.the inhibition studies. Inhibition was carried out at 250 in buffers of pH 5-6, acetate; 6-8 , phosphate; 8-10, Tris. Progress of inhibition was followed by assaying against BOC-Ala-ONp as substrate using a Cary 14 PM recording spectrophotometer equipped with thermostatted cell compartment at 347 nrn.
RESULTS The
effect
of pH upon
butylphosphonate
(ENBP)
AND
the reaction
of elastase
was
in the pH range
studied
dence
of the butyl
compound
was
pound
might
it too fast
to measure
pH-rate
make
profile
for
this
observed
between
presence
of an ionizable
imidazole similar
moiety rate
profile
studied,
inhibition
pH 7. 5 to 9. group
of an histidine with
DISCUSSION
of pKa
pKa
1180
1.
shows
6. 9, which
residue.
an apparent
inhibition
in Fig.
pH profile
ethyl 5.8
at the optimal
is shown The
since
with
Geneste
E-nitrophenyl - 10.
The
npH depen-
by the pentyl pH.
The
bell-shaped
Maximum
inhibition
a dependence
can be iaentified and Bender
of 6. 85 in the hydrolysis
com-
was
on the as of an
(11) reported of the sub-
a
BIOCHEMICAL
Vol. 83, No. 3, 1978
strate,
N-furyl-acryloyl-L-alanine
in rate
of inhibition
either tion
methyl
at higher
a conformational
pH is quite
change
by elastase. This
interesting.
of the active
site
The might
of the enzyme
decrease
be due to or to denatura-
of Inhibition
The
rate
of inhibitor
of inhibition
complex
formation.
The
Where
of elastase
concentration
reversible
between
enzyme pathway
E . I represents
Here
product
with
of inhibition.
higher
than
stant
the total
of elastase
rENBP>
l/kobs
vs.
= 0.
values
The
The
changing
analogous k,/KI
the alkyl
group
for three
to e-propyl,
be 8. 5, 90 and 4 respectively.
bond
inhibitor,
bound
to the enzyme.
and ka is the limiting is considerably
and the value
The
inhibition
kcat/
K m,
alkylphosphonate
It is evident
and
rate
con-
(3)
of KI and FE2were
s-pentyl
of a
by the equation
l/rENBPl
1181
with
concentration
be given
parameter, other
covalent
the pseudo-first-order
respectively.
to the catalytic values
of enzyme
= L t K1 k2 k,r ENBP;
is plotted
2 x 1o-4 M and 4 x 10S3 set -I, KI,
may
the presence
as follows:
irreversibly
concentration,
as a function
preceding
complex
inhibitor
kobs,
inhibition,
2 reveals
of the E * I complex
the initial
enzyme
2, l/kobs
when
Fig.
investigated
be expressed
the inhibitor
l/kobs
In Fig.
may
constant
Since
was
and inhibitor
a noncovalent
KI is the dissociation
rate
by ENBP
at pH 7 and 250.
reaction
E - I is the final
20.
ester,
RESEARCH COMMUNICATIONS
of the enzyme.
Kinetics
W
AND BIOPHYSICAL
and n-hexyl that
of l/KI
computed
= -1 to be
parameter, was
computed
esters
made
were
out of the four
to be by
computed alkylphosphon-
to
Vol. 83, No. 3, 1978
ate esters
BIOCHEMICAL
studied
for
fic inhibitor
is ethyl
100% within
20 minutes
the inhibition R-nitrophenyl
capacity)
is 90.
literature
(12-
using
In recent inhibitors possibility
years
several
in animal
models
that
appropriate potent bitor
inhibitor.
inhibitor for
the
for
the treatment
RESEARCH COMMUNICATIONS
of elastase,
most
This
is higher
various
inhibitors
course
elastase,
(which than
effective
of emphysema
ethyl this
(14-15).
elastase
measure
reported
of
in the
There
of elastase
is a goad by the use of an
n-pentylphosphonate
be used for
studies
be arrested
R-nitrophenyl
of emphysema
is the best
initiated
could
might
It inhibits
any value
have
of the disease
and speci-
of elastase.
investigators
Since
the
n-pentylphosphonate.
and the k,/ KI value
inhibitory
13),
AND BIOPHYSICAL
which
as a powerful at present
is a synthetic
there
inhi-
is no suitable
treatment.
ACKNOWLEDGMENTS This National
investigation Science
Fulbright-Hays
was Foundation.
supported
by grant
P. L. N.
CHE76-14283
acknowledges
a grant
from from
the the
Commission.
REFERENCES 1. 2. 3. 4.
Balo, J. and Banga, I. (1949) Nature, 164, 491. Partridge, S.M. and Davis, H.F. (1955) Biochem. J. 61, 21-30,. Hartley, B.S. (1964) Nature (London) 201, 1284-1287. Walsh, K. and Neurath, H. (1964) Proc. Nat. Acad. Sci. U.S. A. , 52 884-889. Shotton, D. M. and Hartley, B.S. (1970) Nature (London) 225, 802-806. Stroud, R. M. , Kay, L. M. , and.Dickerson, R. E. (1972), Cold Spring Harbor Symp. on &ant. Biol. 36, 125. Shotton, D. M. , and Watson, H. C. (1970) Nature (London) 225, 811-816. Janoff, A. (1972) Am. J. Pathol. 68, 579-591. Kosolapoff, G. M. (1950) Organophosphorus Compounds pp. 146-167, John Wiley and Sons, Inc. , New York. Fukuto, T.R. and Metcalf, R. L. (1959) J. Amer. Chem. Sot., 81, 372-377. Geneste, P. and Bender, M. L. , (1969), Proc. Nat. Acad. Sci. , U. S. A. , 64, 683-685. Visser, L. , Sigman, D. S. , and Blout, E. R. (1971), Biochemistry 10, 735-742. Powers, J. C. and Tuhy, P. M. (1973), Biochemistry 12, 4767-4774. Mittman, C. (1972), Pulmonary Emphysema and Proteolysis, Academic Press, New York, pp. l-537. Power, J. C. (1976) TIBS 1, 211-214.
65: 7. 8. 9. 10. 11. 12. 13. 14. 15.
1182
8lOCHEMlCAl
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
August 14,1978
Pages
ORGANOPHOSPHORUS
COMPOUNDS INHIBITORS
Padma
Received
June
AS ACTIVE
1178-1182
SITE-DIRECTED
OF ELASTASE
L. Nayak and Myron L. Division of Biochemistry Department of Chemistry Northwestern University Evanston, Illinois 60201
Bender
12,1978
SUMMARY Four ethyl R-nitrophenyl alkylphosphonates were studied for the inhibition of elastase. A pH-dependence study using the assay substrate BOC-Ala-ONp or the phosphonate inhibitors showed the participation of an ionizing group with an apparent pKa of 6.9 and a decrease of reaction or inhibition at higher Out of the four compounds investigated ethyl R-nitrophenyl pentylphosPH. phonate was found to be the best inhibitor of elastase as judged from the value which is the measure of inhibitory capacity, is the of k,/ KI. This value, highest reported so far for the inhibition of elastase. INTRODUCTION Elastase
is a pancreatic
(l),
remarkable
similarities
in its
to two
pancreatic
proteolytic
other
three
enzymes
nizes
large
of lysine alanine plete
differ
only
hydrophobic
remarkable almost
structures
degree identical,
containing
and the
process,
similar
geometrical
characterized
of similarity all
catalytic
groups
side
arrangements.
by the presence
sites.
The
binding
trypsin
binding
chains
is similar
0006-291X/78/0833-1178$01.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
to that
1178
recog-
side
residues
the com-
(3-6
).
There
is a They
catalytically
active
sites.
site
which
is acylated
serine,
pockets
and aspartic these adjacent
is hydrophobic of chymotrypsin
chains
especially
determined
enzymes
Furthermore,
of chymotrypsin
These
chymotrypsin
have
of histidine
of binding
and chymotrypsin.
aliphatic
of all three
an active
properties
positively-charged
of workers
in their
possesses
and biological
specificity:
trypsin small
which
trypsin
substrate
chains;
Several
three-dimensional
physical,
enzymes,
and elastase
derivatives.
(2) enzyme
chemical,
in their side
and arginine;
proteolytic
three
acid
are in the
in very
enzymes
are
to the catalytic and open,
the
but contains
a
BIOCHEMICAL
Vol. 83, No. 3, 1978
negatively
charged
elastase idue
aspartate
is narrow
near
A number
of investigations processes
emphysema
(8).
Inhibitors
of functional
elastase.
of primary could
provide
a means
in reality, make upon
use
they
been
shown
have
of studying
of the
both
in vivo.
center
Additionally,
and,
specificity
of valine
res-
be involved
therefore,
inhibitors
as tools their
with
in the attack
a
with
in the investideriva-
inhibitors
in a unique
in reacting
involved
with
radioactive
stoichiometric
mechanism
of purified
stoichiometrically
since
in
in pulmonary
Synthetic
served
these
factors
damage
to react
of the active
catalytic
may
for the characterization
have
the
of a bulky
elastase
tissue
compounds
substrates
same
that
elastic
role
as tags.
are
with
their
structure
be used
suggested
deducing
Organophosphorus
gation tives
have
tool
groups
and the binding
of the presence
a valuable
as for
RESEARCH COMMUNICATIONS
(7).
associated
provide
as well
variety
because
of the cavity
pathological
enzymes
ion in the bottom,
and shallower
the front
AND BIOPHYSICAL
way
can
because,
the enzyme, of the enzyme
substrates. In this
elastase butyl,
communication, with
n-pentyl
four
ethyl
we wish R-nitrophenyl
and n-hexyl),
to report
an account
alkylphosphonates
the phosphorus
analogs
of the inhibition (alkyl
of specific
= q-propyl, substrates
of nof
elastase.
MATERIALS
AND
METHODS
Porcine elastase was a Worthington Biochemical Corp. purified product used without further purification; its substrate BOC-Ala-ONp, was purchased Synthesis of ethyl R-nitrophenyl butylphosphonfrom Vega-Fox Biochemicals. ate (ENBP): Diethyl butyl-phosphonate was prepared by the application of Michaelis-Arbuzov reaction (9) in which equimolar quantities of triethylphosphite and n-butyl bromide were heated under reflux at 1500 to give diethyl butylphosphonate and ethyl bromide. Ethyl butylphosphonochloridate was synthesized by the reaction of diethyl butylphosphonate and phosphorus pentaEthyl R-nitrophenyl butylphosphonate was prepared by chloride in the cold. reacting ethyl butylphosphonochloridate with sodium R-~trophenoxide in toluene according to the method of Fukuto and Metcalf (10). The other three inhibitors which involve a change in the alkyl groups to l-propyl, e-pentyl and n-hexyl were synthesized according to the above procedure. Stock solutions of substrate (Spectra grade, Eastman Kodak
and inhibitor were prepared in acetonitrile Co. ). Buffer solutions were made from
1179
Vol. 83, No. 3, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.25 0.20 0.15 0.1
3
0.05
2 I 5
0
6
7
8
9
4
IO
0
5
x d4 M-’
2
PH
1
pH dependence of the inhibition of elastase by ENBP, 250, E = 2 x 10-6M; I = 1 x 10-4M.;, substrate = BOC-Ala-ONp = 5 x 10s5M. Determination of _t%e inhibition constant _o,f elastase by ENBP at 250, M, I = 0.25 - 2 x 10 M, substrate = BOC-Ala-ONp= pH 7. E = 2 x 10 5 x 1O-5 M.
1.
2.
reagent
grade
chemicals
of ionic
strength,
I - 0.1.
Elastase inhibition. A 25 to 100 fold molar excess of inhibitor over enzyme was used for.the inhibition studies. Inhibition was carried out at 250 in buffers of pH 5-6, acetate; 6-8 , phosphate; 8-10, Tris. Progress of inhibition was followed by assaying against BOC-Ala-ONp as substrate using a Cary 14 PM recording spectrophotometer equipped with thermostatted cell compartment at 347 nrn.
RESULTS The
effect
of pH upon
butylphosphonate
(ENBP)
AND
the reaction
of elastase
was
in the pH range
studied
dence
of the butyl
compound
was
pound
might
it too fast
to measure
pH-rate
make
profile
for
this
observed
between
presence
of an ionizable
imidazole similar
moiety rate
profile
studied,
inhibition
pH 7. 5 to 9. group
of an histidine with
DISCUSSION
of pKa
pKa
1180
1.
shows
6. 9, which
residue.
an apparent
inhibition
in Fig.
pH profile
ethyl 5.8
at the optimal
is shown The
since
with
Geneste
E-nitrophenyl - 10.
The
npH depen-
by the pentyl pH.
The
bell-shaped
Maximum
inhibition
a dependence
can be iaentified and Bender
of 6. 85 in the hydrolysis
com-
was
on the as of an
(11) reported of the sub-
a
BIOCHEMICAL
Vol. 83, No. 3, 1978
strate,
N-furyl-acryloyl-L-alanine
in rate
of inhibition
either tion
methyl
at higher
a conformational
pH is quite
change
by elastase. This
interesting.
of the active
site
The might
of the enzyme
decrease
be due to or to denatura-
of Inhibition
The
rate
of inhibitor
of inhibition
complex
formation.
The
Where
of elastase
concentration
reversible
between
enzyme pathway
E . I represents
Here
product
with
of inhibition.
higher
than
stant
the total
of elastase
rENBP>
l/kobs
vs.
= 0.
values
The
The
changing
analogous k,/KI
the alkyl
group
for three
to e-propyl,
be 8. 5, 90 and 4 respectively.
bond
inhibitor,
bound
to the enzyme.
and ka is the limiting is considerably
and the value
The
inhibition
kcat/
K m,
alkylphosphonate
It is evident
and
rate
con-
(3)
of KI and FE2were
s-pentyl
of a
by the equation
l/rENBPl
1181
with
concentration
be given
parameter, other
covalent
the pseudo-first-order
respectively.
to the catalytic values
of enzyme
= L t K1 k2 k,r ENBP;
is plotted
2 x 1o-4 M and 4 x 10S3 set -I, KI,
may
the presence
as follows:
irreversibly
concentration,
as a function
preceding
complex
inhibitor
kobs,
inhibition,
2 reveals
of the E * I complex
the initial
enzyme
2, l/kobs
when
Fig.
investigated
be expressed
the inhibitor
l/kobs
In Fig.
may
constant
Since
was
and inhibitor
a noncovalent
KI is the dissociation
rate
by ENBP
at pH 7 and 250.
reaction
E - I is the final
20.
ester,
RESEARCH COMMUNICATIONS
of the enzyme.
Kinetics
W
AND BIOPHYSICAL
and n-hexyl that
of l/KI
computed
= -1 to be
parameter, was
computed
esters
made
were
out of the four
to be by
computed alkylphosphon-
to
Vol. 83, No. 3, 1978
ate esters
BIOCHEMICAL
studied
for
fic inhibitor
is ethyl
100% within
20 minutes
the inhibition R-nitrophenyl
capacity)
is 90.
literature
(12-
using
In recent inhibitors possibility
years
several
in animal
models
that
appropriate potent bitor
inhibitor.
inhibitor for
the
for
the treatment
RESEARCH COMMUNICATIONS
of elastase,
most
This
is higher
various
inhibitors
course
elastase,
(which than
effective
of emphysema
ethyl this
(14-15).
elastase
measure
reported
of
in the
There
of elastase
is a goad by the use of an
n-pentylphosphonate
be used for
studies
be arrested
R-nitrophenyl
of emphysema
is the best
initiated
could
might
It inhibits
any value
have
of the disease
and speci-
of elastase.
investigators
Since
the
n-pentylphosphonate.
and the k,/ KI value
inhibitory
13),
AND BIOPHYSICAL
which
as a powerful at present
is a synthetic
there
inhi-
is no suitable
treatment.
ACKNOWLEDGMENTS This National
investigation Science
Fulbright-Hays
was Foundation.
supported
by grant
P. L. N.
CHE76-14283
acknowledges
a grant
from from
the the
Commission.
REFERENCES 1. 2. 3. 4.
Balo, J. and Banga, I. (1949) Nature, 164, 491. Partridge, S.M. and Davis, H.F. (1955) Biochem. J. 61, 21-30,. Hartley, B.S. (1964) Nature (London) 201, 1284-1287. Walsh, K. and Neurath, H. (1964) Proc. Nat. Acad. Sci. U.S. A. , 52 884-889. Shotton, D. M. and Hartley, B.S. (1970) Nature (London) 225, 802-806. Stroud, R. M. , Kay, L. M. , and.Dickerson, R. E. (1972), Cold Spring Harbor Symp. on &ant. Biol. 36, 125. Shotton, D. M. , and Watson, H. C. (1970) Nature (London) 225, 811-816. Janoff, A. (1972) Am. J. Pathol. 68, 579-591. Kosolapoff, G. M. (1950) Organophosphorus Compounds pp. 146-167, John Wiley and Sons, Inc. , New York. Fukuto, T.R. and Metcalf, R. L. (1959) J. Amer. Chem. Sot., 81, 372-377. Geneste, P. and Bender, M. L. , (1969), Proc. Nat. Acad. Sci. , U. S. A. , 64, 683-685. Visser, L. , Sigman, D. S. , and Blout, E. R. (1971), Biochemistry 10, 735-742. Powers, J. C. and Tuhy, P. M. (1973), Biochemistry 12, 4767-4774. Mittman, C. (1972), Pulmonary Emphysema and Proteolysis, Academic Press, New York, pp. l-537. Power, J. C. (1976) TIBS 1, 211-214.
65: 7. 8. 9. 10. 11. 12. 13. 14. 15.
1182
