BIOCHEMICAL
Vol. 79, No. 3, 1977
INACTIVATION
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF CATHEPSIN Bl BY DIAZOMETHYL KETONES Richard
Leary
and Elliott
Shaw
Biology Department Brookhaven NationalLaboratory Upton, New York 11973 Received
October
27,1977
SUMMARY. Benzyloxycarbonyl-phenylalanyl diazomethyl ketone and benzyloxycarbonyl-phenylalanyl-phenylalanyl diazomethyl ketone, which have been shown to inactivate the thiol protease papain by a mechanism different from that of substrate chloromethyl ketone derivatives, have now been examined as inhibitors of cathepsin Bl of beef spleen. The dipeptide derivative irreversibly inactivates this protease rapidly, apparently by affinity labeling. Cathepsin
Bl (E.C.
in many animal logical
tissues
processes.
glucokinase,
lysosomal
1,6
fructose
primary
in
explore
these
and many other
not
a distinction
permit
therefore ketone proteases
derivatives since
(5),
possible
different appeared
initial
observations
Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
subtle
that
growth,
tissue
protease,
class
of reagents
and thiol
cathepsin
a
lack
shown
for (6).
To
selective ketones
do
proteases
was sought.
as potentially
on
and the
chloromethyl
proteases
effects
Bl has recently
of this
directed
indicate
enzymes
may be responsible
serine
promising
unaffected
by observations
proposed
of connective
roles
Protease
between
More
invasive
which
of inactivating
metabolizing
Cathepsin
breakdown
be valuable.
a chemically
(4).
present
of physio-
leaving
suggested
been
detachment,
activity
of extracellular
acid
are
has also
formation
stages
would
It
while
turnover.
activity
in cellular
initial
kinase,
in protein
(3).
shown capable
and amino
on enzymic
collagenolytic
inhibitors
pyruvate
protease
in a number
has been
it
role
metastasis
sulfhydryl
implicated
carbohydrate
biphosphatase
step
to possess
has been
a regulatory
some role
a lysosomal
I
and liver
proteases
Bl may play
)
For example ,
of other
indicating
of
(l),
aldolase,
the activities (3,
3.4.4.-
(7),
Diazcmethyl
selective
for
thiol
of action
on serine
926 ISSN
0006-291
X
BIOCHEMICAL
Vol. 79, No. 3, 1977
proteases
(unpublished
have established inactivators bovine
spleen
that of that
results). Z-Phi
Studies
and Z-Phe-Phil
protease
and examined
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(8), for
its
with
the
thiol
diazomethyl
therefore susceptibility
protease, ketones
cathepsin
papain,
are
effective
B1 was purified
to these
from
inhibitors.
METHODS Reagents: The synthesis of the inhibitors used in this investigation have been previously described (8,9,10). The organomercurial-Sepharose column was prepared by the procedure of Sluyterman and Wijdenes (11). The following chemicals were obtained from commercial sources: Aquasol from New England Nuclear; No-Z-Lysine' nitrophenyl ester from Cycle chemical comfrom Eastman Kodak; Sephadex gels from Pharmacia; pany; mercaptoethanol Bio-Rex 70 from Bio Rad laboratories. Frozen bovine spleens were obtained from Pel-Freeze biochemicals and stored at -20' until needed. Assay: Cathepsin B1 was assayed spectrophotometrically, with No-Z-Lys-ONpl (12), by the addition of enzyme to 2.0 ml of 0.10 M acetate, 0.0025 M mercaptoethanol, 0.001 M EDTA (pH 5.4) followed by the addition of 20 ~1 of 10m2M substrate in 95% acetonitrile. The increase in absorbance at 340 nm was measured. Enzyme activity was expressed as umoles of p-nitrophenol produced min-' ml-l of enzyme solution. The kinetic inactivation experiments were performed at room temperature. The inhibitors, at the appropriate concentration in methanol, were added at zero time to the enzyme solution. The final concentration of methanol was 10%. The extent of enzyme inactivation was determined by assaying an aliquot of the reaction and of a control mixture without inhibitor at various times. Enzyme: Cathepsin Bl was purified from bovine spleen by modification of the procedure of Otto and Risenkoning (13). Supernatent from the spleen extract (Fraction I) from one kilogram of tissue was fractionated with ammonium sulfate and the precipitate collected between 40 and 70% saturation was dissolved in 0.001 M EDTA and dialyzed overnight versus 8 liters of this solution. Dialysates from three batches were combined (Fraction II) and applied to a Bio-Rex 70 column (3.5 x 60 cm) previously equilibrated with 0.09 M citrate, 0.001 M EDTA (pH 5.4) buffer, and the column was developed with the same The fractions with high cathepsin B1 activity buffer. were pooled and concentrated with an Amicon ultrafiltration apparatus, equipped with a DM-10 membrane. The concentrated pool (Fraction III) fran the Bio-Rex 70 column was applied to a SephadexG-75column (2.0 x 100 cm) developed with 0.09 M citrate, 0.001 M EDTA (pH 5.4) buffer and fractions with high specific activity were pooled (Fraction IV). Aliquots of this material were dialyzed overnight versus 0.10 M acetate, 0.10 M NaCl, 0.001 M EDTA (pH 5.4) buffer and applied to an organomercurial Sepharose column (10 ml). Unadsorbed proteins were washed from the column with 0.10 M acetate, 0.10 M NaCl, 0.001 M EDTA (pH 5.4). Absorbed proteins were eluted from the column with the same buffer containing 0.01 M cysteine, and fractions containing significant amounts of protein were pooled for use (Fraction V) in the inactivation experiments described. All the steps outlined in the purification procedure, with of the initial acid extraction and the organanercurial Sepharose f 2= lAbbreviations: benzyloxycarbonyl, -0Np = p-nitrophenylester.
927
the exception chromato-
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The final specific activity of the cathepsin graphy were carried out at 4". B obtained as outlined above, ranged from 14.13 to 26.95 poles of nitrop tl en01 released per minute per A280 per ml of enzyme solution in four different preparations of the enzyme. When this material was subjected to disc gel electrophoresis at pH 4.5 (13) followed by staining for amidase activity (14) a single band of color was observed although Coumassie blue revealed the presence of other proteins in addition. A summary of one preparation is shown below: Fraction
Total Units
Vol.
I II III IV V
5030 260 5.5 32 39
ml ml ml ml
Spec.
5229 3489 1284 894 713
ml
Act.
Purification Factor
Recovery
1 37 167 392 523
100% 67% 25% 17% 14%
0.027 1.00 4.52 10.62 14.13
HESULTS AND DISCUSSION The incubation in
a gradual
permitted
loss
of activity
the demonstration
the enzyme with
different,
with
chloromethyl
that
No-Z-L-Phe
the
in this agents
inactivation
with
orientation the chemical
of the potential
of the
inhibitor
This
inhibitors of the
excess
is
ester
complex binding
active
essentially
928
is site
inert
This (8)
site. with
mercaptoethanol This
fact
a competitive
suggest employed
of these to provide
of cathepsin diazomethyl
B1
ketones
formation thought
and the inhibitor
by cathepsin
Bl by the diazomethyl
from prior
1).
in comparison
the
nitrophenyl
was
in the case of papain
ketone
at the
for
kinetics (Figure
experiments.
initial
action
was strikingly
order
binding
with
diazomethyl
results
the enzyme.
first
inactivation
of cathepsin
investigation
pseudo
unreactive
combine
of NCY-Z-L-Lys
slow
inhibitor
Z-Phe-PheCHN2,
observed
chemically
during
This
was a competitive
of an extended
and do not
in the buffer
of the hydrolysis that
was also
are
x 10m4M> resulted
Kk = 8.0 x 10m4M.
concentrations
ketones
ketones
observation
Z-Phe-CHN2
(2.5
173 minutes.
derivative,
the participation
Diazomethyl
50% in
following
in reactivity
and reflects
Z-Phe-CHN2
used with
inactivation
micromolar
increase
present
that
of the dipeptide
rapid
observed
B1 with reaching
the substrate
The action
marked
of cathepsin
rean
B1 such that ketone
function
Vol. 79, No. 3, 1977
ol
’
0
’ 6
’
BIOCHEMICAL
’
’
12 TIME
01
’
’
18 (min)
’
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
’
24
30 TIME
bin)
Fig. 1. Inactivation of cathepsin Bl by Z-Phe-PheCHN2 at pH 5.4 in 0.09 M acetate, 0.0020 M mercaptoethanol, 0.0009 M EDTA containing 10% acetonitrile at room temperature. Reaction mixture (2 ml) contained 40 pl of enzyme, specific activity 19.5. Aliquots (200 ~1) removed at indicated times for assay. Fig. ditions
2. Inactivation of cathepsin as in legend to Fig. 1.
is expressed kinetic Bl is
and alkylation
characteristics expected
activation.
sites
the biological
methyl
to substantiate
in
out,
inactivation pH dependencies.
with
follows in the It
this
a different case of papain
was not
929
possible
work
on the
ketones
portion
with
as well
(8)
shown,
to examine
reagent
character
of
as indicate
of protease mechanism
cathepsin
of the in-
of the
on the
center class
Con-
character
the peptide
Bl active
attainable
the reaction
of diazomethyl
to shed some light
in the cathepsin
potency
ketone
contrasting
variations
ketones.
Continuing
the affinity-labeling
way and can be expected
the binding
As pointed
of the enzyme results. of the reaction
Structural
are under
Bl by chloromethyl
inhibitor. than for this
chloroexample, with
by
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE INACTIVATION
I
OF CATHEPSIN Bl BY CHLORO- and DIAZO-METHYL
REAGENT
CONCENTRATION (M)
t+(min)
kl(min-')
KETONES mol -1 )
kl,I(min-l
No-Tos-LysCH2Cl
5
x 10 -6
3
0.23
46,200
No-Tos-PheCH2Cl
5
x 10 -5
5.3
0.131
2,610
No-Z-PheCH2C1
2
x 10 -6
7.25
0.096
48,000
No-Z-PheCHN2
2.5 x 1O-4
173
0.004
No-Z-Phe-PhecHN2
1.5 x 1o-6
21
0.033
22,000
0.11
22,000
5.0 x 1o-6
cathepsin
B1 due to instability
ination
of diazomethyl
source
of error
the rates
some basis
and Z-PheCH2C1
were
type
to follow pseudo
rates first
ethanol,
linear
a rate
(15).
with
of protein a particular
reaction
in this
most
of cathepsin reported
chemistry amino
of chloromethyl
acid
likely
Bl with
curve
reaction
the various study
However, other
930
an attempt not
used; with
sulfhydryl
The
kl,[I1
(last
(8). and
investigated
assigned previous
mercapto-
in calculating
I with
been
follow
the reagent.
chloromethyl
has not
residue.
cathepsin
did
of reactivity
be definitely
with
Tos-PheCH2C1
without
in Table
a
of reagents,
was utilized
and cannot
ketones
two types
was reacting
is
(8).
concentrations
comparison
in this
of study
by Barrett
2, the
are presented for
ketones
of human liver
observed
inactivation
Data
Contam-
B1 by Tos-Lys-CH2C1,
at the reagent
of the
type
of the
As shown in Figure
as a basis
ketones
by chloromethyl
comparison
had been
in excess, part
above neutrality.
The inactivation
kinetics
The reaction
level
for
of reagent
presented
diazaaethyl
against
of cathepsin
of inactivation.
column)
preparations
measured.
order
present
initial
ketone
of inactivation
B1 by this
of the enzyme
to be guarded
To provide
6.4
16.1
at the
to the reaction
studies proteases
of the (16)
Vol. 79, No. 3, 1977
have definitely the active
BIOCHEMICAL
established center
cysteine
L-Phe-diazomethyl active that
ketone
center the
ketones
Peptide
(17),
diazomethyl
and --in vitro
physiological
results
an acid
(8),
thus
it
apparently
ketone
derivatives
and pathological
a similar site
requiring appear
of the role processes
of papain
the alkylation
the esterification
a reaction
alkylation
seems reasonable
by active
from
investigations
from
Bl follows
protease,
to result
from
and the inactivation
was shown to result
of cathepsin
has been shown groups
inactivation
residue
residue
of pepsin,
carboxyl
--in vivo
cysteine
inactivation
activation
that
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in which
by No-Cbzof the
to assume
course.
The in-
directed
diazomethyl
of active cupric to be well
of cathepsin
of
ion
site activation.
suited Bl in
for the
the enzyme has been
implicated. ACKNOWLEDGMENTS This research carried out at Brookhaven National Laboratory, under the auspices of the United States Energy Research and Development Administration and by the United States Public Health Service Grant BM-17849. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Greenbaum, K. M. (1971) in The Enzymes (Boyer, P. D., ed.) pp. 475-483, Academic Press, New York. Otto, K. (1971) in Tissue Proteinases (Barrett, A. J., and Dingle, J. T., eds.) pp. l-28, North Holland Publishing Company, Amsterdam. Pontremoli, S., DeFlora, A., Salamino, F., Melloni, E., and Horecker, B. L. (1975) Proc. Natl. Acad. Sci. USA, 72, 2969-2973. Sylven, B. (1970) in Chemotherapy of Cancer Dissemination and Metastasis (Garattini, S., and Franchi, G., eds.) pp. 129-138, Raven Press, New York. Burleigh, M. C., Barrett, A. J., and Lazarus, G. S. (1974) Biochem. J ., 137, 387-398. Holtzman, E. (1975) Lysosomes: A Survey, pp. 110-115 and 179-184, Springer-Verlag, New York. Shaw, E. (1970) Physiol. Rev., 50, 244-296. Leary, R., Larsen, D., Watanabe, H., and Shaw, E., in press, Biochemistry. Shaw, E. (1967) in Methods in Enzymology, Vol. 11 (Hirs, C. H. W., ed.) Shaw, E., and Glover, G. (1970) Arch. Biochem. Biophys., 139, 298-305. Sluyterman, L. A. AE., and Wijdenes, J. (1970) Biochim. Biophys. Acta, 200, 593-595. Bajkowski, A. S., and Frankfater, A. (1975) Anal. Biochem., 68, 119-127. Otto, K,, and Risenkoning, H. (1975) Biochim. Biophys. Acta, 379, 462-475. Barrett, A. J. (1972) Anal. Biochem., 47, 280-293. Barrett, A. J. (1973) Biochem. J., 131, 809-822. Glazer, A. N., and Smith, E. L. (1971) in The Enzymes, Vol. III (Boyer, P. D., ed.) pp. 502-546, Academic Press, New York. Fry, K. T., Kim, O.-K., Spona, J., and Hamilton, G. A. (1970) Biochemistry, 9, 4624-4632.
931
Vol. 79, No. 3, 1977
INACTIVATION
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF CATHEPSIN Bl BY DIAZOMETHYL KETONES Richard
Leary
and Elliott
Shaw
Biology Department Brookhaven NationalLaboratory Upton, New York 11973 Received
October
27,1977
SUMMARY. Benzyloxycarbonyl-phenylalanyl diazomethyl ketone and benzyloxycarbonyl-phenylalanyl-phenylalanyl diazomethyl ketone, which have been shown to inactivate the thiol protease papain by a mechanism different from that of substrate chloromethyl ketone derivatives, have now been examined as inhibitors of cathepsin Bl of beef spleen. The dipeptide derivative irreversibly inactivates this protease rapidly, apparently by affinity labeling. Cathepsin
Bl (E.C.
in many animal logical
tissues
processes.
glucokinase,
lysosomal
1,6
fructose
primary
in
explore
these
and many other
not
a distinction
permit
therefore ketone proteases
derivatives since
(5),
possible
different appeared
initial
observations
Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
subtle
that
growth,
tissue
protease,
class
of reagents
and thiol
cathepsin
a
lack
shown
for (6).
To
selective ketones
do
proteases
was sought.
as potentially
on
and the
chloromethyl
proteases
effects
Bl has recently
of this
directed
indicate
enzymes
may be responsible
serine
promising
unaffected
by observations
proposed
of connective
roles
Protease
between
More
invasive
which
of inactivating
metabolizing
Cathepsin
breakdown
be valuable.
a chemically
(4).
present
of physio-
leaving
suggested
been
detachment,
activity
of extracellular
acid
are
has also
formation
stages
would
It
while
turnover.
activity
in cellular
initial
kinase,
in protein
(3).
shown capable
and amino
on enzymic
collagenolytic
inhibitors
pyruvate
protease
in a number
has been
it
role
metastasis
sulfhydryl
implicated
carbohydrate
biphosphatase
step
to possess
has been
a regulatory
some role
a lysosomal
I
and liver
proteases
Bl may play
)
For example ,
of other
indicating
of
(l),
aldolase,
the activities (3,
3.4.4.-
(7),
Diazcmethyl
selective
for
thiol
of action
on serine
926 ISSN
0006-291
X
BIOCHEMICAL
Vol. 79, No. 3, 1977
proteases
(unpublished
have established inactivators bovine
spleen
that of that
results). Z-Phi
Studies
and Z-Phe-Phil
protease
and examined
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(8), for
its
with
the
thiol
diazomethyl
therefore susceptibility
protease, ketones
cathepsin
papain,
are
effective
B1 was purified
to these
from
inhibitors.
METHODS Reagents: The synthesis of the inhibitors used in this investigation have been previously described (8,9,10). The organomercurial-Sepharose column was prepared by the procedure of Sluyterman and Wijdenes (11). The following chemicals were obtained from commercial sources: Aquasol from New England Nuclear; No-Z-Lysine' nitrophenyl ester from Cycle chemical comfrom Eastman Kodak; Sephadex gels from Pharmacia; pany; mercaptoethanol Bio-Rex 70 from Bio Rad laboratories. Frozen bovine spleens were obtained from Pel-Freeze biochemicals and stored at -20' until needed. Assay: Cathepsin B1 was assayed spectrophotometrically, with No-Z-Lys-ONpl (12), by the addition of enzyme to 2.0 ml of 0.10 M acetate, 0.0025 M mercaptoethanol, 0.001 M EDTA (pH 5.4) followed by the addition of 20 ~1 of 10m2M substrate in 95% acetonitrile. The increase in absorbance at 340 nm was measured. Enzyme activity was expressed as umoles of p-nitrophenol produced min-' ml-l of enzyme solution. The kinetic inactivation experiments were performed at room temperature. The inhibitors, at the appropriate concentration in methanol, were added at zero time to the enzyme solution. The final concentration of methanol was 10%. The extent of enzyme inactivation was determined by assaying an aliquot of the reaction and of a control mixture without inhibitor at various times. Enzyme: Cathepsin Bl was purified from bovine spleen by modification of the procedure of Otto and Risenkoning (13). Supernatent from the spleen extract (Fraction I) from one kilogram of tissue was fractionated with ammonium sulfate and the precipitate collected between 40 and 70% saturation was dissolved in 0.001 M EDTA and dialyzed overnight versus 8 liters of this solution. Dialysates from three batches were combined (Fraction II) and applied to a Bio-Rex 70 column (3.5 x 60 cm) previously equilibrated with 0.09 M citrate, 0.001 M EDTA (pH 5.4) buffer, and the column was developed with the same The fractions with high cathepsin B1 activity buffer. were pooled and concentrated with an Amicon ultrafiltration apparatus, equipped with a DM-10 membrane. The concentrated pool (Fraction III) fran the Bio-Rex 70 column was applied to a SephadexG-75column (2.0 x 100 cm) developed with 0.09 M citrate, 0.001 M EDTA (pH 5.4) buffer and fractions with high specific activity were pooled (Fraction IV). Aliquots of this material were dialyzed overnight versus 0.10 M acetate, 0.10 M NaCl, 0.001 M EDTA (pH 5.4) buffer and applied to an organomercurial Sepharose column (10 ml). Unadsorbed proteins were washed from the column with 0.10 M acetate, 0.10 M NaCl, 0.001 M EDTA (pH 5.4). Absorbed proteins were eluted from the column with the same buffer containing 0.01 M cysteine, and fractions containing significant amounts of protein were pooled for use (Fraction V) in the inactivation experiments described. All the steps outlined in the purification procedure, with of the initial acid extraction and the organanercurial Sepharose f 2= lAbbreviations: benzyloxycarbonyl, -0Np = p-nitrophenylester.
927
the exception chromato-
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The final specific activity of the cathepsin graphy were carried out at 4". B obtained as outlined above, ranged from 14.13 to 26.95 poles of nitrop tl en01 released per minute per A280 per ml of enzyme solution in four different preparations of the enzyme. When this material was subjected to disc gel electrophoresis at pH 4.5 (13) followed by staining for amidase activity (14) a single band of color was observed although Coumassie blue revealed the presence of other proteins in addition. A summary of one preparation is shown below: Fraction
Total Units
Vol.
I II III IV V
5030 260 5.5 32 39
ml ml ml ml
Spec.
5229 3489 1284 894 713
ml
Act.
Purification Factor
Recovery
1 37 167 392 523
100% 67% 25% 17% 14%
0.027 1.00 4.52 10.62 14.13
HESULTS AND DISCUSSION The incubation in
a gradual
permitted
loss
of activity
the demonstration
the enzyme with
different,
with
chloromethyl
that
No-Z-L-Phe
the
in this agents
inactivation
with
orientation the chemical
of the potential
of the
inhibitor
This
inhibitors of the
excess
is
ester
complex binding
active
essentially
928
is site
inert
This (8)
site. with
mercaptoethanol This
fact
a competitive
suggest employed
of these to provide
of cathepsin diazomethyl
B1
ketones
formation thought
and the inhibitor
by cathepsin
Bl by the diazomethyl
from prior
1).
in comparison
the
nitrophenyl
was
in the case of papain
ketone
at the
for
kinetics (Figure
experiments.
initial
action
was strikingly
order
binding
with
diazomethyl
results
the enzyme.
first
inactivation
of cathepsin
investigation
pseudo
unreactive
combine
of NCY-Z-L-Lys
slow
inhibitor
Z-Phe-PheCHN2,
observed
chemically
during
This
was a competitive
of an extended
and do not
in the buffer
of the hydrolysis that
was also
are
x 10m4M> resulted
Kk = 8.0 x 10m4M.
concentrations
ketones
ketones
observation
Z-Phe-CHN2
(2.5
173 minutes.
derivative,
the participation
Diazomethyl
50% in
following
in reactivity
and reflects
Z-Phe-CHN2
used with
inactivation
micromolar
increase
present
that
of the dipeptide
rapid
observed
B1 with reaching
the substrate
The action
marked
of cathepsin
rean
B1 such that ketone
function
Vol. 79, No. 3, 1977
ol
’
0
’ 6
’
BIOCHEMICAL
’
’
12 TIME
01
’
’
18 (min)
’
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
’
24
30 TIME
bin)
Fig. 1. Inactivation of cathepsin Bl by Z-Phe-PheCHN2 at pH 5.4 in 0.09 M acetate, 0.0020 M mercaptoethanol, 0.0009 M EDTA containing 10% acetonitrile at room temperature. Reaction mixture (2 ml) contained 40 pl of enzyme, specific activity 19.5. Aliquots (200 ~1) removed at indicated times for assay. Fig. ditions
2. Inactivation of cathepsin as in legend to Fig. 1.
is expressed kinetic Bl is
and alkylation
characteristics expected
activation.
sites
the biological
methyl
to substantiate
in
out,
inactivation pH dependencies.
with
follows in the It
this
a different case of papain
was not
929
possible
work
on the
ketones
portion
with
as well
(8)
shown,
to examine
reagent
character
of
as indicate
of protease mechanism
cathepsin
of the in-
of the
on the
center class
Con-
character
the peptide
Bl active
attainable
the reaction
of diazomethyl
to shed some light
in the cathepsin
potency
ketone
contrasting
variations
ketones.
Continuing
the affinity-labeling
way and can be expected
the binding
As pointed
of the enzyme results. of the reaction
Structural
are under
Bl by chloromethyl
inhibitor. than for this
chloroexample, with
by
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE INACTIVATION
I
OF CATHEPSIN Bl BY CHLORO- and DIAZO-METHYL
REAGENT
CONCENTRATION (M)
t+(min)
kl(min-')
KETONES mol -1 )
kl,I(min-l
No-Tos-LysCH2Cl
5
x 10 -6
3
0.23
46,200
No-Tos-PheCH2Cl
5
x 10 -5
5.3
0.131
2,610
No-Z-PheCH2C1
2
x 10 -6
7.25
0.096
48,000
No-Z-PheCHN2
2.5 x 1O-4
173
0.004
No-Z-Phe-PhecHN2
1.5 x 1o-6
21
0.033
22,000
0.11
22,000
5.0 x 1o-6
cathepsin
B1 due to instability
ination
of diazomethyl
source
of error
the rates
some basis
and Z-PheCH2C1
were
type
to follow pseudo
rates first
ethanol,
linear
a rate
(15).
with
of protein a particular
reaction
in this
most
of cathepsin reported
chemistry amino
of chloromethyl
acid
likely
Bl with
curve
reaction
the various study
However, other
930
an attempt not
used; with
sulfhydryl
The
kl,[I1
(last
(8). and
investigated
assigned previous
mercapto-
in calculating
I with
been
follow
the reagent.
chloromethyl
has not
residue.
cathepsin
did
of reactivity
be definitely
with
Tos-PheCH2C1
without
in Table
a
of reagents,
was utilized
and cannot
ketones
two types
was reacting
is
(8).
concentrations
comparison
in this
of study
by Barrett
2, the
are presented for
ketones
of human liver
observed
inactivation
Data
Contam-
B1 by Tos-Lys-CH2C1,
at the reagent
of the
type
of the
As shown in Figure
as a basis
ketones
by chloromethyl
comparison
had been
in excess, part
above neutrality.
The inactivation
kinetics
The reaction
level
for
of reagent
presented
diazaaethyl
against
of cathepsin
of inactivation.
column)
preparations
measured.
order
present
initial
ketone
of inactivation
B1 by this
of the enzyme
to be guarded
To provide
6.4
16.1
at the
to the reaction
studies proteases
of the (16)
Vol. 79, No. 3, 1977
have definitely the active
BIOCHEMICAL
established center
cysteine
L-Phe-diazomethyl active that
ketone
center the
ketones
Peptide
(17),
diazomethyl
and --in vitro
physiological
results
an acid
(8),
thus
it
apparently
ketone
derivatives
and pathological
a similar site
requiring appear
of the role processes
of papain
the alkylation
the esterification
a reaction
alkylation
seems reasonable
by active
from
investigations
from
Bl follows
protease,
to result
from
and the inactivation
was shown to result
of cathepsin
has been shown groups
inactivation
residue
residue
of pepsin,
carboxyl
--in vivo
cysteine
inactivation
activation
that
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in which
by No-Cbzof the
to assume
course.
The in-
directed
diazomethyl
of active cupric to be well
of cathepsin
of
ion
site activation.
suited Bl in
for the
the enzyme has been
implicated. ACKNOWLEDGMENTS This research carried out at Brookhaven National Laboratory, under the auspices of the United States Energy Research and Development Administration and by the United States Public Health Service Grant BM-17849. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Greenbaum, K. M. (1971) in The Enzymes (Boyer, P. D., ed.) pp. 475-483, Academic Press, New York. Otto, K. (1971) in Tissue Proteinases (Barrett, A. J., and Dingle, J. T., eds.) pp. l-28, North Holland Publishing Company, Amsterdam. Pontremoli, S., DeFlora, A., Salamino, F., Melloni, E., and Horecker, B. L. (1975) Proc. Natl. Acad. Sci. USA, 72, 2969-2973. Sylven, B. (1970) in Chemotherapy of Cancer Dissemination and Metastasis (Garattini, S., and Franchi, G., eds.) pp. 129-138, Raven Press, New York. Burleigh, M. C., Barrett, A. J., and Lazarus, G. S. (1974) Biochem. J ., 137, 387-398. Holtzman, E. (1975) Lysosomes: A Survey, pp. 110-115 and 179-184, Springer-Verlag, New York. Shaw, E. (1970) Physiol. Rev., 50, 244-296. Leary, R., Larsen, D., Watanabe, H., and Shaw, E., in press, Biochemistry. Shaw, E. (1967) in Methods in Enzymology, Vol. 11 (Hirs, C. H. W., ed.) Shaw, E., and Glover, G. (1970) Arch. Biochem. Biophys., 139, 298-305. Sluyterman, L. A. AE., and Wijdenes, J. (1970) Biochim. Biophys. Acta, 200, 593-595. Bajkowski, A. S., and Frankfater, A. (1975) Anal. Biochem., 68, 119-127. Otto, K,, and Risenkoning, H. (1975) Biochim. Biophys. Acta, 379, 462-475. Barrett, A. J. (1972) Anal. Biochem., 47, 280-293. Barrett, A. J. (1973) Biochem. J., 131, 809-822. Glazer, A. N., and Smith, E. L. (1971) in The Enzymes, Vol. III (Boyer, P. D., ed.) pp. 502-546, Academic Press, New York. Fry, K. T., Kim, O.-K., Spona, J., and Hamilton, G. A. (1970) Biochemistry, 9, 4624-4632.
931
