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On the activation of trypsinogen. A study of peptide models related to the N-terminal sequence of the zymogen
Vol. 29, No. 2, 1967
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ON THE ACTIVATION OF TRYPSINOGEN. A STUDY OF PEPTIDE MODELS RELATED TO THE N-TERMINAL SEQUENCE OF THE ZYMOGEN. Institut
M.Delaage, P.Desnuelle, M.Lazdunski Biologique, Facultd des Sciences,Marseille,France E.Bricas, J.Savrda de Biochimie, Faculte des Sciences, Orsay, France.
de Chirnie Institut
Received September 21, 1967 The N-terminal vation (1955)) goire
peptides
of bovine
(Davie
porcine
(Charles
and al.
(1966))
Asp-Asp-Asp-Asp first
attributed
to this
and Lazdunski on the
cribed.
The kinetic
N-terminal
in
In
tryptic
in
and ovine
cormnon the
(Lazdunski
and Delaage
the
specificity
of several
of bovine
Tg,
(Bricteux-Gr&
the
is hydroly-
roles
influence
for
and Fabre
bond which Several
paper
the acti-
sequence
activation.
this
of
Desnuelle
the Lys-Ile
behavior
sequence
the course
(1955), (1963))
have
the
sequence
1967).
residues
and al.
preceding
step
in
and Neurath
Tg all
just
zed as the
liberated
might
1967, of the
Lys-Ile
peptides
bond related
be
Delaage aspartyl is
des-
to the
Val-Asp4-Lys-Ile-Val-Gly,
is
studied. Methods 1. Their
The peptides
:
purity
chromatography leucine
has been
The kinetic
(Savrda
been obtained
checked
on silica
aminopeptidase.
bed elsewhere
have
gel
by electrophoresis,
and by enzymatic
Details
of their
and Bricas
parameters
-----_--__--_---__--___I____D______ The following abbreviations sin ; ChTg A, Chymotrypsinogen
as sumarized
in Figure thin
degradation
synthesis
will
layer with be descri-
in preparation).
for
the
hydrolysis
are
used A.
: Tg,
235
by trypsin Trypsinogen
of ; Ti,
the Try&+
Vol. 29, No. 2, 1967
Val
BIOCHEMICAL
Asp
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Ile
Asp
Val
GUY
Z
IV Val
Ala
Ala
ZtoH zIt% H--OEt Y \"-I 1 ow
z
Val
GUY
BOG
OEt z
OEt
Z Z
Ile
LYS
'
OBut
/ H /Boc
N3
OBut OBut V
Z
Fig. 1 During peptide bond synthesis, the carboxyl groups were protected by conversion to the t-butyl ester (-OBut) or the ethyl The a- and C-amino groups were protected with benester (-OEt). zyloxycarbonyl (Z-) and t-butyloxycarbonyl (BOG) respectively. For the formation of peptide bonds the carboxyl groups (-OH) of the Z-aminoacids were activated either by conversion into the the N-hydroxysuccinimide ester (-OSU) p-nitrophenyl ester (-ONP), or the acid azide (N3-) or were activated by the coupling agents dicyclohexylcarbodiimide (DCC) or carbonic mixed anhydride (MA). At each step of the synthesis the Z-protecting group was removed by catalytic hydrogenation and the free "amino component" (H-) was coupled to the following carboxyl activated Z- aminoacid or -OBut and BOG were removed by treatment with trifluopeptide. roacetic acid.
Lys-Ile direct rated
bond
in
evaluation protons
for
bovine
of
activation
Ti
the
peptides
by the
were obtained
pH-stat
at pH 9 (fig. between
I
BOC
the
of ChTg A in
technique
2 A).
of
(2) A study
peptides the
by two methods. the number of
the
of
236
of peptides
libe-
competition
and ChTg A. The initial
presence
(1) A
is
rate :
Vol. 29, No. 2, 1967
v2 =
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
v2 K1 S2/K2 S1 + Ki
with K'1 =Kl
(l+
-1
s2 K2
where V2, K2 (1.2 x 10B3M at pH 8.0, Sl are respectively concentrations Sl (fig.
1" C) and S2 ; VI' Kl and the maximal rates, Michaelis constants and
of ChTg A and the peptides.
2 B) gives
A plot
of l/v2
against
Ki and then K,.
0
c P
b)
0
x
A/ 1
-X-Y
f
x10-2
Fig. 2 A Lineweaver-Burk determination of Vl and Kl for peptide (IV) without (a) or with Ca2-t 65 mM (b), pH 9.0, 25', ionic strength 0.3. v is expressed in )unole of peptide hydrolysed/min/mg Ti. Fig. 2 B Determination of Vl and Kl by competition for Ti between ChTg A and peptide (I) (a) or ChTg A and peptide (IV) (b), ionic strength 0.1. v2 is expressed in chymotrypsin units (with N-acetyl-L-tyrosine ethyl ester as a substrate)/min/mg Ti.
Results
and Discussion
:
and II
and of peptides
III
In table
and IV shows that slight
of Kl are accompanied by large variations
I, a comparison
changes in kcat.
of peptides variations
Conversely,
of Kl are accompanied in the case of peptides
by small changes in kcat.
These independent 237
I
large IV and V
changes of Kl and kcat
Vol. 29, No. 2, 1967
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE
Ga2+
Peptides Lys-Ile-Val-Gly
suggest
~1-1 8.0
that
and that
of
active
serine
The results of aspartyl mation
of
0 (cl 0.39 (c) effect
(a) 1 positive
2.51
0.46 (c) effect
(a) (b) (b)
2.8 3.6
(VI
0 0 50
.0255 .0905 .0905
(a) (b) (b)
1.45 7.0 7.0
25',
pB 9.0
constants
is
the
in
trypsin
shown in
residues
(a) (a) no
(set";
1.92 3.35 1.11
; (b)
kcat
4.25 8.53
kcat
0 0 65
the Michaelis
tants,
(mM)
(IV) -Lys-Ile-Val-Gly
l",
the
(III)
-Lys-Ile-Val-Gly
Val-(Ala)2
Kl
0 50 0 50
(II)
:Asp)2-Lys-Ile-Val-Gly Val-(Asp)2
(mN)
0
(I)
Asp-Lys-Ile-Val-Gly
(a)
I
near
the Michaelis
rate
the
are
l",
(a) (b) (b)
pH 9.0.
true
equilibrium
constant
k2 for
I indicate
that
x
Table
; (c)
(b) (b)
the
consacylation
0
Lys-Ile
complex.
bond does
the
accumulation
not
favor
(1) A comparison
of
the
the K 1 for
-------_--_---------------------------x The mechanism
of
action
of Ti
involves
k3 )ES2----+E complex, k cat
n
andkel,k dently.
ES -" k2+k3 cat
the
+ P2
+ pl acylated complex ;KPI(S
=k 29K=-
on Ser
the , 183.
following ES1 is
238
the Michaelis
V - kcat
k-l+%! - k3 and KS = k2+k3 kl 'rl and these parameters
fc1
steps
l
[Eog;
When k2
indepen-
for-
Vol. 29, No. 2, 1967
peptides
BIOCHEMICAL
I, II,
independent
III
and IV shows that
the binding
substrate
for
residues.
residues
trypsin,
2.5.
different
effect sites
V increases
as N-benzoyl-L-argininamide.
of Ca2+ in the activation of inert
of the strategic
This double
in peptide
its K1 and kcat are much more favourable
on the formation
of hydrolysis
(2) The replace-
Peptide V, is an excellent
the usual amide substrates
tant effect
is largely
75 times at lo pH 8 and 37 times at 25' pH 9
by a factor
The presence
binding
by alanyl
constant,
k =at is increased
hydrolysis
this constant
of the number of aspartyl
ment of these residues
than for
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
proteins
and on the rate
bond Lys(6)-Ile
is due to the binding
(Delaage
medium has an impor-
and Lazdunski
(7) of bovine
of the cation (1967)).
Tg.
at two
The effect
on the
of the Lys (6). Ile (7) bond is apparently due to the of Ca2+ on the 4 aspartyl residues. The rate of hydroly-
sis of the Lys-Ile
bond is increased by a factor of about 7 for Tg in 5 x 10-2 M Ca2+ (Mc Donald and Kunitz (1941)) and
native
an analysis
of the results
the initial
rate
S-sulfo
of the hydrolysis
Tg is also increased
tions.
This Ca2+ effect
fluence
on the spatial
with
of Pechere and al.
that conclusion
the hydrolysis
6 to 7 times under these same condi-
structure we find
shows that
of the same bond in denatured
appears then to be independent
of peptide
of any in-
of the zymogen. In accordance
a very similar
effect
of Ca2-l- on
IV where 6.5 x 10-2 M Ca2' (at this
concentration
the effect
and increases
k cat by a factor
at low substrate
(1958),
is maximum) lowers K1 by a factor
concentration
of 1.3 thus increasing (S1 << K1) by a factor
239
of 3
the rate of 3.9.
Vol. 29, No. 2, 1967
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The Ca2+ effect two aspartyl
residues.
'cent observation tion
only appears with
blocked
Our results
that calcium
of a Tg derivative (Radhakrishnan
peptides
III
are in agreement with
is not necessary
in which the carboxylate and al.
and IV which have
for
the re-
the activagroups are
(1967)).
Financial help by Delegation G&n&ale a la Recherche Scientifique et Technique (Convention 66.00.056), Centre National de la Recherche Scientifique (RCP 23) and National Institute of Health (Grant AM 04642) is gratefully acknowledged. References Bricteux-Gregoire, S., Schynd, R. and Florkin, M., Biochim.Biophys. Acta 127, 277 (1966). Charles, M., Rovery, M., Guidoni, A. and Desnuelle, P., Biochim. Biophys.Acta 69, 115 (1963). Davie, E.W. and Neurath, H., J.Biol.Chem., 212, 818 (1955). Delaage, M. and Lazdunski M., Biochem.Biophys.Research Comm.,28, 390 (1967). Desnuelle, P. and Fabre, C., Biochim.Biophys.Acta, 18, 29 (1955). Mc Donald, M.R. and Kunitz, M., J.Gen.Physiol., 25, 53 (1941). Gabeloteau, C. and Desnuelle, P., Arch.Biochem.Biophys., 69, 475 (1957). Lazdunski,M. and Delaage,M., Biochim.Biophys.Acta, 140, 417 (1967). Pechbre, J.F., Dixon, G.M., Maybury, R.M. and Neurath, H., J.Biol. Chem., 233, 1364 (1958). Radhakrishnan, T.M., Walsh,K.A. and Neurath, H., J.Am.Chem.Soc., 89, 3059 (1967).
240
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ON THE ACTIVATION OF TRYPSINOGEN. A STUDY OF PEPTIDE MODELS RELATED TO THE N-TERMINAL SEQUENCE OF THE ZYMOGEN. Institut
M.Delaage, P.Desnuelle, M.Lazdunski Biologique, Facultd des Sciences,Marseille,France E.Bricas, J.Savrda de Biochimie, Faculte des Sciences, Orsay, France.
de Chirnie Institut
Received September 21, 1967 The N-terminal vation (1955)) goire
peptides
of bovine
(Davie
porcine
(Charles
and al.
(1966))
Asp-Asp-Asp-Asp first
attributed
to this
and Lazdunski on the
cribed.
The kinetic
N-terminal
in
In
tryptic
in
and ovine
cormnon the
(Lazdunski
and Delaage
the
specificity
of several
of bovine
Tg,
(Bricteux-Gr&
the
is hydroly-
roles
influence
for
and Fabre
bond which Several
paper
the acti-
sequence
activation.
this
of
Desnuelle
the Lys-Ile
behavior
sequence
the course
(1955), (1963))
have
the
sequence
1967).
residues
and al.
preceding
step
in
and Neurath
Tg all
just
zed as the
liberated
might
1967, of the
Lys-Ile
peptides
bond related
be
Delaage aspartyl is
des-
to the
Val-Asp4-Lys-Ile-Val-Gly,
is
studied. Methods 1. Their
The peptides
:
purity
chromatography leucine
has been
The kinetic
(Savrda
been obtained
checked
on silica
aminopeptidase.
bed elsewhere
have
gel
by electrophoresis,
and by enzymatic
Details
of their
and Bricas
parameters
-----_--__--_---__--___I____D______ The following abbreviations sin ; ChTg A, Chymotrypsinogen
as sumarized
in Figure thin
degradation
synthesis
will
layer with be descri-
in preparation).
for
the
hydrolysis
are
used A.
: Tg,
235
by trypsin Trypsinogen
of ; Ti,
the Try&+
Vol. 29, No. 2, 1967
Val
BIOCHEMICAL
Asp
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Ile
Asp
Val
GUY
Z
IV Val
Ala
Ala
ZtoH zIt% H--OEt Y \"-I 1 ow
z
Val
GUY
BOG
OEt z
OEt
Z Z
Ile
LYS
'
OBut
/ H /Boc
N3
OBut OBut V
Z
Fig. 1 During peptide bond synthesis, the carboxyl groups were protected by conversion to the t-butyl ester (-OBut) or the ethyl The a- and C-amino groups were protected with benester (-OEt). zyloxycarbonyl (Z-) and t-butyloxycarbonyl (BOG) respectively. For the formation of peptide bonds the carboxyl groups (-OH) of the Z-aminoacids were activated either by conversion into the the N-hydroxysuccinimide ester (-OSU) p-nitrophenyl ester (-ONP), or the acid azide (N3-) or were activated by the coupling agents dicyclohexylcarbodiimide (DCC) or carbonic mixed anhydride (MA). At each step of the synthesis the Z-protecting group was removed by catalytic hydrogenation and the free "amino component" (H-) was coupled to the following carboxyl activated Z- aminoacid or -OBut and BOG were removed by treatment with trifluopeptide. roacetic acid.
Lys-Ile direct rated
bond
in
evaluation protons
for
bovine
of
activation
Ti
the
peptides
by the
were obtained
pH-stat
at pH 9 (fig. between
I
BOC
the
of ChTg A in
technique
2 A).
of
(2) A study
peptides the
by two methods. the number of
the
of
236
of peptides
libe-
competition
and ChTg A. The initial
presence
(1) A
is
rate :
Vol. 29, No. 2, 1967
v2 =
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
v2 K1 S2/K2 S1 + Ki
with K'1 =Kl
(l+
-1
s2 K2
where V2, K2 (1.2 x 10B3M at pH 8.0, Sl are respectively concentrations Sl (fig.
1" C) and S2 ; VI' Kl and the maximal rates, Michaelis constants and
of ChTg A and the peptides.
2 B) gives
A plot
of l/v2
against
Ki and then K,.
0
c P
b)
0
x
A/ 1
-X-Y
f
x10-2
Fig. 2 A Lineweaver-Burk determination of Vl and Kl for peptide (IV) without (a) or with Ca2-t 65 mM (b), pH 9.0, 25', ionic strength 0.3. v is expressed in )unole of peptide hydrolysed/min/mg Ti. Fig. 2 B Determination of Vl and Kl by competition for Ti between ChTg A and peptide (I) (a) or ChTg A and peptide (IV) (b), ionic strength 0.1. v2 is expressed in chymotrypsin units (with N-acetyl-L-tyrosine ethyl ester as a substrate)/min/mg Ti.
Results
and Discussion
:
and II
and of peptides
III
In table
and IV shows that slight
of Kl are accompanied by large variations
I, a comparison
changes in kcat.
of peptides variations
Conversely,
of Kl are accompanied in the case of peptides
by small changes in kcat.
These independent 237
I
large IV and V
changes of Kl and kcat
Vol. 29, No. 2, 1967
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE
Ga2+
Peptides Lys-Ile-Val-Gly
suggest
~1-1 8.0
that
and that
of
active
serine
The results of aspartyl mation
of
0 (cl 0.39 (c) effect
(a) 1 positive
2.51
0.46 (c) effect
(a) (b) (b)
2.8 3.6
(VI
0 0 50
.0255 .0905 .0905
(a) (b) (b)
1.45 7.0 7.0
25',
pB 9.0
constants
is
the
in
trypsin
shown in
residues
(a) (a) no
(set";
1.92 3.35 1.11
; (b)
kcat
4.25 8.53
kcat
0 0 65
the Michaelis
tants,
(mM)
(IV) -Lys-Ile-Val-Gly
l",
the
(III)
-Lys-Ile-Val-Gly
Val-(Ala)2
Kl
0 50 0 50
(II)
:Asp)2-Lys-Ile-Val-Gly Val-(Asp)2
(mN)
0
(I)
Asp-Lys-Ile-Val-Gly
(a)
I
near
the Michaelis
rate
the
are
l",
(a) (b) (b)
pH 9.0.
true
equilibrium
constant
k2 for
I indicate
that
x
Table
; (c)
(b) (b)
the
consacylation
0
Lys-Ile
complex.
bond does
the
accumulation
not
favor
(1) A comparison
of
the
the K 1 for
-------_--_---------------------------x The mechanism
of
action
of Ti
involves
k3 )ES2----+E complex, k cat
n
andkel,k dently.
ES -" k2+k3 cat
the
+ P2
+ pl acylated complex ;KPI(S
=k 29K=-
on Ser
the , 183.
following ES1 is
238
the Michaelis
V - kcat
k-l+%! - k3 and KS = k2+k3 kl 'rl and these parameters
fc1
steps
l
[Eog;
When k2
indepen-
for-
Vol. 29, No. 2, 1967
peptides
BIOCHEMICAL
I, II,
independent
III
and IV shows that
the binding
substrate
for
residues.
residues
trypsin,
2.5.
different
effect sites
V increases
as N-benzoyl-L-argininamide.
of Ca2+ in the activation of inert
of the strategic
This double
in peptide
its K1 and kcat are much more favourable
on the formation
of hydrolysis
(2) The replace-
Peptide V, is an excellent
the usual amide substrates
tant effect
is largely
75 times at lo pH 8 and 37 times at 25' pH 9
by a factor
The presence
binding
by alanyl
constant,
k =at is increased
hydrolysis
this constant
of the number of aspartyl
ment of these residues
than for
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
proteins
and on the rate
bond Lys(6)-Ile
is due to the binding
(Delaage
medium has an impor-
and Lazdunski
(7) of bovine
of the cation (1967)).
Tg.
at two
The effect
on the
of the Lys (6). Ile (7) bond is apparently due to the of Ca2+ on the 4 aspartyl residues. The rate of hydroly-
sis of the Lys-Ile
bond is increased by a factor of about 7 for Tg in 5 x 10-2 M Ca2+ (Mc Donald and Kunitz (1941)) and
native
an analysis
of the results
the initial
rate
S-sulfo
of the hydrolysis
Tg is also increased
tions.
This Ca2+ effect
fluence
on the spatial
with
of Pechere and al.
that conclusion
the hydrolysis
6 to 7 times under these same condi-
structure we find
shows that
of the same bond in denatured
appears then to be independent
of peptide
of any in-
of the zymogen. In accordance
a very similar
effect
of Ca2-l- on
IV where 6.5 x 10-2 M Ca2' (at this
concentration
the effect
and increases
k cat by a factor
at low substrate
(1958),
is maximum) lowers K1 by a factor
concentration
of 1.3 thus increasing (S1 << K1) by a factor
239
of 3
the rate of 3.9.
Vol. 29, No. 2, 1967
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The Ca2+ effect two aspartyl
residues.
'cent observation tion
only appears with
blocked
Our results
that calcium
of a Tg derivative (Radhakrishnan
peptides
III
are in agreement with
is not necessary
in which the carboxylate and al.
and IV which have
for
the re-
the activagroups are
(1967)).
Financial help by Delegation G&n&ale a la Recherche Scientifique et Technique (Convention 66.00.056), Centre National de la Recherche Scientifique (RCP 23) and National Institute of Health (Grant AM 04642) is gratefully acknowledged. References Bricteux-Gregoire, S., Schynd, R. and Florkin, M., Biochim.Biophys. Acta 127, 277 (1966). Charles, M., Rovery, M., Guidoni, A. and Desnuelle, P., Biochim. Biophys.Acta 69, 115 (1963). Davie, E.W. and Neurath, H., J.Biol.Chem., 212, 818 (1955). Delaage, M. and Lazdunski M., Biochem.Biophys.Research Comm.,28, 390 (1967). Desnuelle, P. and Fabre, C., Biochim.Biophys.Acta, 18, 29 (1955). Mc Donald, M.R. and Kunitz, M., J.Gen.Physiol., 25, 53 (1941). Gabeloteau, C. and Desnuelle, P., Arch.Biochem.Biophys., 69, 475 (1957). Lazdunski,M. and Delaage,M., Biochim.Biophys.Acta, 140, 417 (1967). Pechbre, J.F., Dixon, G.M., Maybury, R.M. and Neurath, H., J.Biol. Chem., 233, 1364 (1958). Radhakrishnan, T.M., Walsh,K.A. and Neurath, H., J.Am.Chem.Soc., 89, 3059 (1967).
240