Vol. 84, No. 1, 1978 September
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages
14, 1978
ACYL AND AMINO INTERMZDIATES
IN REACTIONS
95-101
CATALYZED
BY THERMOLYSIN H. Tsuzuki
K. Morihara, Shionogi
Research
Laboratory,
Fukushima-ku, Received
July
and T. Oka Shionogi
Osaka
553,
& Co.,
Ltd.,
Japan
25,1978 SUMMARY
Thermolysin showed peculiar transpeptidation reactions. Leu-Leu and/or Leu-Leu-Leu were produced at ca. pH 7 from Leu-Leu-NH2 and Cbz-Leu-Leu. Isotope experiments indicated that the transpeptidation products did not use leucine reWith Leu-Trp-Met, leased from the substrates as an acceptor. Leu-Leu-Leu and Met-Met were produced as transLeu-Leu, A comparative study was done with apeptidation products. chymotrypsin and pepsin. These results would indicate that thermolysin catalyzed reactions proceed via both acyl and amino intermediates depending upon the substrates, which has been proposed for the mechanism of pepsin. This may also be true in some cases for chymotrypsin and other proteases, which have been known as enzymes of the acyl-enzyme mechanism. The acyl-enzyme
mechanism
established
(1).
thermolysin
has been
which
Glu-143,
of a water peptide
is
on the
donates
or amino
promotes of
somewhat to the
of
the
(2) in the
attack
scissile
depolarized
peptide
However,
pathway
by
by
nitrogen
no information
catalysis
via
the
acyl
intermediate.
Wang and Hofmann transpeptidation intermediates
been
is well
study
carbon
a proton
the
X-ray
base,
intermediate.
concerning
proteases of catalysis
its
carbonyl
has already
a tetrahedral
available
from
as a general
molecule
Zn2+ , and His-231 forming
proposed
acting
serine
a mechanism
Recently,
bond which
for
(3) have
reactions depending
shown that
which upon
proceed
the
pig via
substrates.
pepsin acyl
catalyzes
and amino
Leu-Leu
and/or
0006-291X/78/0841-0095$01.00/0 95
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 84, No. 1, 1978
Leu-Leu-Leu transfer which
BIOCHEMICAL
were produced type)
and from
leucine-CO-E
was postulated
form
or Cbz-Phe-Leu-Leu mediately With
either
substrate,
the
Leu-Leu-NH2
from
enzyme)
substrate
(amino-transfer or E-NH-leucine
Leu-Leu-Leu-NH2
into
Leu-Trp-Met acyl
the
(3) or
type), (amino
which
above-mentioned
would
in enzyme)
im-
products.
[14 C]Leu-Tyr-[3H]Leu
and amino
(acyl-
and Leu-Leu-Leu-Leu-NH2
and Cbz-Phe-Leu-Leu-Leu,
be hydrolyzed
both
RESEARCH COMMUNICATIONS
Cbz-Phe-Leu
(acyl to
AND BIOPHYSICAL
transpeptidation
(4) products
as a were
formed. In the
present
study,
undertaken
to investigate
way of reactions
catalyzed
by thermolysin,
we determined
transpeptidation
reactions
using
substrates.
MATERIALS Thermolysin (crystals) three times) were supplied and Worthington Biochemical respectively.
Hofmann's
the
paththe
AND METHODS
and a-chymotrypsin from Daiwa Kasei Corp., Freehold,
(crystallized Co., Osaka, Japan N.J., U.S.A.,
&-Leucine, L-methionine, Cbz-Gly-Leu and Cbz-Leu-Leu were supplied frgm The Protein Research Foundation, Minoh, Japan. Leu-Leu, Leu-Leu-Leu, Met-Met, Cbz-Phe-Leu and LeuLeu-NH2 were purchased from Vega-Fox Biochemicals, Tucson, Leu-Trp-Met was obtained from Research Plus AZ., U.S.A. Laboratories Inc., Denville, N.J., U.S.A. [14C]Leucine (354 mCi/mmol) was from The Radiochemical Centre, Amersham, Abbreviated designations of amino acid derivaBucks., U.K. tives, peptides, or their derivatives conform to the tentative rules of the IUPAC-IUB Commission on Biochemical Nomenclature Except when specified, the constituent amino acids (1972). were all of the &-configuration. The quantitative determination of amino acids and oligopeptides in the reaction mixture was made using an amino acid analyzer (Japan Electron Optics Laboratory, Model JLC-3) The mixture according to the method of Wang and Hofmann (3). of Leu-NH2 and Leu-Leu-NH2 was separated on a column (0.8 x 15 cm) of chromosorv #2611 resin using 0.7 M sodium citrate, The elution pH 5.28, at 55'C. The flow rate was 70 ml/h. and Leu-Leu-NH2 was 193 and 225 min, retime of Leu-NH T2e mixture of the other reaction products was spectively. separated on a column (0.3 x 50 cm) of chromosorv #3105 resin at 60°C. The buffers were 0.2 M sodium citrate, pH 4.25, and change occurred 0.35 M sodium citrate, pH 5.28, and the buffer at 70 min. The elution time of G-methionine, &-leucine, Met-
96
BIOCHEMICAL
Vol. 84, No. 1, 1978
I
2
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3
4
0
Time
I
2
3
4
( h1
Fig. 1. Progress curve of cleavage of Cbz-Leu-Leu (2 mM) and Leu-Leu-NH2 (2 mM) by thermolysin (1 mg/ml) at pH 7.1 and at 37'C. The products were analyzed by an amino acid analyzer. 0, Leucine; 0, Leu-Leu; (3Leu-Leu-Leu; and X, Leu-NH2.
Met, Leu-Leu respectively.
and Leu-Leu-Leu
were 74,
82,
142,
152 and 164 min,
The reaction products from substrates were separated and identified by high-voltage electrophoresis in pyridine-acetic acid-water (1:20:279, by volume) at pH 3.5 and 25 V/cm for 120 min using authentic compounds for comparison. For the analysis of reaction mixtures containing [14C]leucine, guide strips were stained and the unstained part of the electrophoretogram was cut into 2 cm-wide bands. These were put into scintillation bottles containing p-bis-(o-methylstyryl)benzene (0.05%) and 2,5-diphenyloxazole (0.5%) in toluene, and their radioactivity was measured in a Beckmann DPM-100 liquid-scintillation counter.
RESULTS AND DISCUSSION The progress
of
and Leu-Leu-NH 2 is leucine
in either
leucine,
Leu-Leu
the
sum of
the action
of thermolysin
shown in Fig. peptide
is
1.
the products
is
The C- or N-terminal
converted
and Leu-Leu-Leu.
on Cbz-Leu-Leu
into
the products,
In the case of Leu-Leu-NH2,
almost
97
equal
to Leu-NH2,
which
Vol. 84, No. 1, 1978
BIOCHEMICAL
I.
Table
Effect
transpeptidation
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of pH on the hydrolysis
reactions
catalyzed
and
by thermolysin
The reaction mixture contained 2 mMsubstrate, thermolysin
(1 mg/ml), 0.1 M Tris-buffer
as indicated
(various pH values
in the table) and 1 mMCaC12, which was kept
at 37OCfor 2 h. Substrate
Leucine Leu-Leu Leu-Leu-Leu Leu-NH2 (mol of leucine/lOO mol of substrate)
pH
6.2
26
9.3
5 0.4
17 28 15 2
6.2
3 5
19
1
25
3 1
11 1
3 4
Cbz-Leu-Leu 7.1 7. 8
10
Leu-Leu-NH2 7.1 7,8 9.3
supports
this
is -ca.
7,
as shown in Table
The transpeptidation with
those
Table
II.
The data
tion the
occurs
AA-Leu
is
of pig
and pepsin,
pepsin
were
In thermolysin
hydrolysis
very
Leu-NH2,
slow
in comparison
can catalyze
as well
instead
Lou-Leu-NH2
ia comparable
peptidation
activity
with
that
Lt?u-Leu-NH2;
to that
of pepsin
the
98
reverse
is
case can be
reaction
with
Cbz-
of thermolysin. reaction the
velocity
of thermolysin.
roughly
study
reactions
Similar the
the
of transpeptida-
transpeptidation
as with
from
while
although
com-
as seen in
catalyzed
or glycine.
a-chymotrypsin,
were
deduced
when AA is &-phenylalanine,
a-Chymotrypsin
with
of thermolysin
of a-chymotrypsin
preferential
with
transpeptidation
I.
case when AA is &-leucine
observed
33 38 30 4
0
reactions
of Wang and Hofmann (3). of Cbz-AA-Leu,
7 2
The optimum pH for
proposal.
pared
9 14
corresponds
of
The trans-
to that
of
8lOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 84, No. 1, 1978
Table II.
Comparison
of transpeptidation
with The reaction (pH 7.1),
trypsin
those by a-chymotrypsin
mixture
contained
Enzyme
The reaction
Cbz-Phe-Leu Cbz-Leu-Leu Leu-Leu-NH2 Leu-NH2
a-Chymotrypsin
the concentration
20 20 4 20
1.5 14 3 3
38
53 2 0.8 15
5 15 7 18
0 0 15 2
20 60
3 12 0.5 2
17 32 1 2.5
6 10
2 5 2 4
a Reference
(3):
[S] = 2 ml-f, [E] = 1 mg/ml,
pH 4.7,
37°C.
b Reference
(3):
[S] = 4 mM, [E] = 2 mg/ml,
pH 3.4,
37°C.
Similar
Leu-Trp-Met
results
further
in
mixture. indicates
leucine
crystal
respects
that
the
of
structure to
the
the
from
The mechanism the
as seen
of
acid of
of
are
in
these
Table
enzymes
III.
[14C]leucine
summarized
transpeptidation substrates
in
in
does
was the
Table
not
IV,
occur
using
is
based
on
similar
in
as an acceptor.
proteases penicillopepsin,
thermolysin
in
and a-chymotrypsin
presence
The results that
released
observed
by thermolysin
determined
reaction
were
as substrate,
Transpeptidation
which
of a-chymo-
4 28 36 25
Pepsin
thermolysin.
(2 mgiml)
74 10 12 5
Cbz-Phe-Leua
with
0.1 M Tris-buffer
Leucine Leu-Leu Leu-Leu-Leu Leu-NH2 (mol of leucine/lOO mol of substrate)
Cbz-Phe-Leu Cbz-Leu-Leu Cbz-Gly-Leu Leu-Leu-NH2
Thermolysin
and pepsin
time is shown in the table.
Reaction time (h)
Substrate
by thermolysin
(1 mg/ml> or a-chymotrypsin
In the case of Leu-NH2,
was 10 mglml.
catalyzed
2 mM substrate,
1 mM CaC12, and thennolysin
was kept at 37'C.
reactions
(2).
99
(5),
which is
Transpeptidation
many is
a
Vol. 84, No. 1, 1978
Table
III.
BIOCHEMICAL
Transpeptidation
AND BIOPHYSICAL
reactions
of Leu-Trp-Met
o-chymotrypsin The reaction (pH 7.1), which
mixture
1 mM CaC12,
was kept
contained
was 2 or 20 hours,
Thermolysin
3
a-Chymotrypsin
2
Pepsina
3
Leu-Leu (mol
[S]
IV.
3
2
71
3
12
36
2
4
= 2 mM, [E]
The other
in Table
= 0.6 mg/ml,
(0.5
ml)
37'C,
2 h.
[14C]leucine
contained
0.2 mM [14C]-
conditions
are
II. Product
Enzyme
9
catalyzed
of
experimental
Met-Met-Met
25
pH 3.4,
or a-chymotrypsin
in the presence mixture
(1 pCi>.
radioactivity
Reactions
Leu-Leu and/or Leu-Leu-Leu
Leucine 182
Background
Thermolysin
or a-chymotrypsin
6
Thermolysin
Each reaction
described
of thermolysin
(2 mg/ml),
8
transpeptidation
leucine
or a-chymotrypsin
Leu-Leu-Leu Methionine Met-Met of leucine/lOOmolof substrate)
28
(3):
Table
time
0.1 M Tris-buffer
respectively.
Leucine
a Reference
(1 mg/ml)
The reaction
by thermolysin,
and pepsin
2 mM Leu-Trp-Met,
and thermolysin
at 37°C.
RESEARCH COMMUNICATIONS
I
Cbz-Leu-Leu + [14C]leucine
(c.p.m.) Leu-NH2
141
84
73,580
139
76
60,382
186
89
143
103
136
70
Leu-Leu-NH2 + [14C]leucine
Chymotrypsin
Cbz-Leu-Leu + [14C]leucine Leu-NH2 + [14C]leucine
74,449 / 71,581
100
Vol. 84, No. 1, 1978
BIOCHEMICAL
phenomenon peculiar
to
both
acyl
AND BIOPHYSICAL
the acid
proteases,
and amino intermediates
(2).
in this
study
general
base mechanism of hydrolysis
the
with
existence
thermolysin.
of a ternary
and amino fragment bond in place
of
an amino-enzyme lease
being
for
and other
determined
thermolysin
work. his
with
acyl
fragment peptide
an anhydride
the order
of
affinity binding
and in some cases
(5) assuming
susceptible
either
a
product
re-
of the
acyl
sites. for
or
This may
chymotrypsin
have been known as enzymes of
the
mechanism.
University We also
technical
of
respective
which
Acknowledgment. Toronto
enzyme,
via
observed
pepsin,
proposed
of the
by the relative
to their
proteases
acyl-enzyme
involvement
intermediate,
or amino moiety be true
the
cleavage
proceeds
was also
case of
is of
and This
In the
complex
after
RESEARCH COMMUNICATIONS
We wish to thank for
thank
helpful to Dr.
assistance
Professor
suggestions M. Takagi
in isotope
of
T. Hofmann of
concerning this
laboratory
this for
experiment.
REFERENCES 1) 2) 3) 4) 5)
Blow, D. M. (1976) Act. Chem. Res. 2, 145-152. Kester, W. R., and Matthews, B-W. (1977) Biochemistry 5, 2506-2516 . Wang, T. T., and Hofmann, T. (1976) Biochem. J. 153, 691-699. Newmark, A. K., and Knowles, J. R. (1975) J. Am. Chem. sot. 97, 3557-3559. James, M. N. G., HSU, I-N., and Delbaere, T. J. (1977) Nature 267, 808-813.
101
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages
14, 1978
ACYL AND AMINO INTERMZDIATES
IN REACTIONS
95-101
CATALYZED
BY THERMOLYSIN H. Tsuzuki
K. Morihara, Shionogi
Research
Laboratory,
Fukushima-ku, Received
July
and T. Oka Shionogi
Osaka
553,
& Co.,
Ltd.,
Japan
25,1978 SUMMARY
Thermolysin showed peculiar transpeptidation reactions. Leu-Leu and/or Leu-Leu-Leu were produced at ca. pH 7 from Leu-Leu-NH2 and Cbz-Leu-Leu. Isotope experiments indicated that the transpeptidation products did not use leucine reWith Leu-Trp-Met, leased from the substrates as an acceptor. Leu-Leu-Leu and Met-Met were produced as transLeu-Leu, A comparative study was done with apeptidation products. chymotrypsin and pepsin. These results would indicate that thermolysin catalyzed reactions proceed via both acyl and amino intermediates depending upon the substrates, which has been proposed for the mechanism of pepsin. This may also be true in some cases for chymotrypsin and other proteases, which have been known as enzymes of the acyl-enzyme mechanism. The acyl-enzyme
mechanism
established
(1).
thermolysin
has been
which
Glu-143,
of a water peptide
is
on the
donates
or amino
promotes of
somewhat to the
of
the
(2) in the
attack
scissile
depolarized
peptide
However,
pathway
by
by
nitrogen
no information
catalysis
via
the
acyl
intermediate.
Wang and Hofmann transpeptidation intermediates
been
is well
study
carbon
a proton
the
X-ray
base,
intermediate.
concerning
proteases of catalysis
its
carbonyl
has already
a tetrahedral
available
from
as a general
molecule
Zn2+ , and His-231 forming
proposed
acting
serine
a mechanism
Recently,
bond which
for
(3) have
reactions depending
shown that
which upon
proceed
the
pig via
substrates.
pepsin acyl
catalyzes
and amino
Leu-Leu
and/or
0006-291X/78/0841-0095$01.00/0 95
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 84, No. 1, 1978
Leu-Leu-Leu transfer which
BIOCHEMICAL
were produced type)
and from
leucine-CO-E
was postulated
form
or Cbz-Phe-Leu-Leu mediately With
either
substrate,
the
Leu-Leu-NH2
from
enzyme)
substrate
(amino-transfer or E-NH-leucine
Leu-Leu-Leu-NH2
into
Leu-Trp-Met acyl
the
(3) or
type), (amino
which
above-mentioned
would
in enzyme)
im-
products.
[14 C]Leu-Tyr-[3H]Leu
and amino
(acyl-
and Leu-Leu-Leu-Leu-NH2
and Cbz-Phe-Leu-Leu-Leu,
be hydrolyzed
both
RESEARCH COMMUNICATIONS
Cbz-Phe-Leu
(acyl to
AND BIOPHYSICAL
transpeptidation
(4) products
as a were
formed. In the
present
study,
undertaken
to investigate
way of reactions
catalyzed
by thermolysin,
we determined
transpeptidation
reactions
using
substrates.
MATERIALS Thermolysin (crystals) three times) were supplied and Worthington Biochemical respectively.
Hofmann's
the
paththe
AND METHODS
and a-chymotrypsin from Daiwa Kasei Corp., Freehold,
(crystallized Co., Osaka, Japan N.J., U.S.A.,
&-Leucine, L-methionine, Cbz-Gly-Leu and Cbz-Leu-Leu were supplied frgm The Protein Research Foundation, Minoh, Japan. Leu-Leu, Leu-Leu-Leu, Met-Met, Cbz-Phe-Leu and LeuLeu-NH2 were purchased from Vega-Fox Biochemicals, Tucson, Leu-Trp-Met was obtained from Research Plus AZ., U.S.A. Laboratories Inc., Denville, N.J., U.S.A. [14C]Leucine (354 mCi/mmol) was from The Radiochemical Centre, Amersham, Abbreviated designations of amino acid derivaBucks., U.K. tives, peptides, or their derivatives conform to the tentative rules of the IUPAC-IUB Commission on Biochemical Nomenclature Except when specified, the constituent amino acids (1972). were all of the &-configuration. The quantitative determination of amino acids and oligopeptides in the reaction mixture was made using an amino acid analyzer (Japan Electron Optics Laboratory, Model JLC-3) The mixture according to the method of Wang and Hofmann (3). of Leu-NH2 and Leu-Leu-NH2 was separated on a column (0.8 x 15 cm) of chromosorv #2611 resin using 0.7 M sodium citrate, The elution pH 5.28, at 55'C. The flow rate was 70 ml/h. and Leu-Leu-NH2 was 193 and 225 min, retime of Leu-NH T2e mixture of the other reaction products was spectively. separated on a column (0.3 x 50 cm) of chromosorv #3105 resin at 60°C. The buffers were 0.2 M sodium citrate, pH 4.25, and change occurred 0.35 M sodium citrate, pH 5.28, and the buffer at 70 min. The elution time of G-methionine, &-leucine, Met-
96
BIOCHEMICAL
Vol. 84, No. 1, 1978
I
2
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3
4
0
Time
I
2
3
4
( h1
Fig. 1. Progress curve of cleavage of Cbz-Leu-Leu (2 mM) and Leu-Leu-NH2 (2 mM) by thermolysin (1 mg/ml) at pH 7.1 and at 37'C. The products were analyzed by an amino acid analyzer. 0, Leucine; 0, Leu-Leu; (3Leu-Leu-Leu; and X, Leu-NH2.
Met, Leu-Leu respectively.
and Leu-Leu-Leu
were 74,
82,
142,
152 and 164 min,
The reaction products from substrates were separated and identified by high-voltage electrophoresis in pyridine-acetic acid-water (1:20:279, by volume) at pH 3.5 and 25 V/cm for 120 min using authentic compounds for comparison. For the analysis of reaction mixtures containing [14C]leucine, guide strips were stained and the unstained part of the electrophoretogram was cut into 2 cm-wide bands. These were put into scintillation bottles containing p-bis-(o-methylstyryl)benzene (0.05%) and 2,5-diphenyloxazole (0.5%) in toluene, and their radioactivity was measured in a Beckmann DPM-100 liquid-scintillation counter.
RESULTS AND DISCUSSION The progress
of
and Leu-Leu-NH 2 is leucine
in either
leucine,
Leu-Leu
the
sum of
the action
of thermolysin
shown in Fig. peptide
is
1.
the products
is
The C- or N-terminal
converted
and Leu-Leu-Leu.
on Cbz-Leu-Leu
into
the products,
In the case of Leu-Leu-NH2,
almost
97
equal
to Leu-NH2,
which
Vol. 84, No. 1, 1978
BIOCHEMICAL
I.
Table
Effect
transpeptidation
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of pH on the hydrolysis
reactions
catalyzed
and
by thermolysin
The reaction mixture contained 2 mMsubstrate, thermolysin
(1 mg/ml), 0.1 M Tris-buffer
as indicated
(various pH values
in the table) and 1 mMCaC12, which was kept
at 37OCfor 2 h. Substrate
Leucine Leu-Leu Leu-Leu-Leu Leu-NH2 (mol of leucine/lOO mol of substrate)
pH
6.2
26
9.3
5 0.4
17 28 15 2
6.2
3 5
19
1
25
3 1
11 1
3 4
Cbz-Leu-Leu 7.1 7. 8
10
Leu-Leu-NH2 7.1 7,8 9.3
supports
this
is -ca.
7,
as shown in Table
The transpeptidation with
those
Table
II.
The data
tion the
occurs
AA-Leu
is
of pig
and pepsin,
pepsin
were
In thermolysin
hydrolysis
very
Leu-NH2,
slow
in comparison
can catalyze
as well
instead
Lou-Leu-NH2
ia comparable
peptidation
activity
with
that
Lt?u-Leu-NH2;
to that
of pepsin
the
98
reverse
is
case can be
reaction
with
Cbz-
of thermolysin. reaction the
velocity
of thermolysin.
roughly
study
reactions
Similar the
the
of transpeptida-
transpeptidation
as with
from
while
although
com-
as seen in
catalyzed
or glycine.
a-chymotrypsin,
were
deduced
when AA is &-phenylalanine,
a-Chymotrypsin
with
of thermolysin
of a-chymotrypsin
preferential
with
transpeptidation
I.
case when AA is &-leucine
observed
33 38 30 4
0
reactions
of Wang and Hofmann (3). of Cbz-AA-Leu,
7 2
The optimum pH for
proposal.
pared
9 14
corresponds
of
The trans-
to that
of
8lOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 84, No. 1, 1978
Table II.
Comparison
of transpeptidation
with The reaction (pH 7.1),
trypsin
those by a-chymotrypsin
mixture
contained
Enzyme
The reaction
Cbz-Phe-Leu Cbz-Leu-Leu Leu-Leu-NH2 Leu-NH2
a-Chymotrypsin
the concentration
20 20 4 20
1.5 14 3 3
38
53 2 0.8 15
5 15 7 18
0 0 15 2
20 60
3 12 0.5 2
17 32 1 2.5
6 10
2 5 2 4
a Reference
(3):
[S] = 2 ml-f, [E] = 1 mg/ml,
pH 4.7,
37°C.
b Reference
(3):
[S] = 4 mM, [E] = 2 mg/ml,
pH 3.4,
37°C.
Similar
Leu-Trp-Met
results
further
in
mixture. indicates
leucine
crystal
respects
that
the
of
structure to
the
the
from
The mechanism the
as seen
of
acid of
of
are
in
these
Table
enzymes
III.
[14C]leucine
summarized
transpeptidation substrates
in
in
does
was the
Table
not
IV,
occur
using
is
based
on
similar
in
as an acceptor.
proteases penicillopepsin,
thermolysin
in
and a-chymotrypsin
presence
The results that
released
observed
by thermolysin
determined
reaction
were
as substrate,
Transpeptidation
which
of a-chymo-
4 28 36 25
Pepsin
thermolysin.
(2 mgiml)
74 10 12 5
Cbz-Phe-Leua
with
0.1 M Tris-buffer
Leucine Leu-Leu Leu-Leu-Leu Leu-NH2 (mol of leucine/lOO mol of substrate)
Cbz-Phe-Leu Cbz-Leu-Leu Cbz-Gly-Leu Leu-Leu-NH2
Thermolysin
and pepsin
time is shown in the table.
Reaction time (h)
Substrate
by thermolysin
(1 mg/ml> or a-chymotrypsin
In the case of Leu-NH2,
was 10 mglml.
catalyzed
2 mM substrate,
1 mM CaC12, and thennolysin
was kept at 37'C.
reactions
(2).
99
(5),
which is
Transpeptidation
many is
a
Vol. 84, No. 1, 1978
Table
III.
BIOCHEMICAL
Transpeptidation
AND BIOPHYSICAL
reactions
of Leu-Trp-Met
o-chymotrypsin The reaction (pH 7.1), which
mixture
1 mM CaC12,
was kept
contained
was 2 or 20 hours,
Thermolysin
3
a-Chymotrypsin
2
Pepsina
3
Leu-Leu (mol
[S]
IV.
3
2
71
3
12
36
2
4
= 2 mM, [E]
The other
in Table
= 0.6 mg/ml,
(0.5
ml)
37'C,
2 h.
[14C]leucine
contained
0.2 mM [14C]-
conditions
are
II. Product
Enzyme
9
catalyzed
of
experimental
Met-Met-Met
25
pH 3.4,
or a-chymotrypsin
in the presence mixture
(1 pCi>.
radioactivity
Reactions
Leu-Leu and/or Leu-Leu-Leu
Leucine 182
Background
Thermolysin
or a-chymotrypsin
6
Thermolysin
Each reaction
described
of thermolysin
(2 mg/ml),
8
transpeptidation
leucine
or a-chymotrypsin
Leu-Leu-Leu Methionine Met-Met of leucine/lOOmolof substrate)
28
(3):
Table
time
0.1 M Tris-buffer
respectively.
Leucine
a Reference
(1 mg/ml)
The reaction
by thermolysin,
and pepsin
2 mM Leu-Trp-Met,
and thermolysin
at 37°C.
RESEARCH COMMUNICATIONS
I
Cbz-Leu-Leu + [14C]leucine
(c.p.m.) Leu-NH2
141
84
73,580
139
76
60,382
186
89
143
103
136
70
Leu-Leu-NH2 + [14C]leucine
Chymotrypsin
Cbz-Leu-Leu + [14C]leucine Leu-NH2 + [14C]leucine
74,449 / 71,581
100
Vol. 84, No. 1, 1978
BIOCHEMICAL
phenomenon peculiar
to
both
acyl
AND BIOPHYSICAL
the acid
proteases,
and amino intermediates
(2).
in this
study
general
base mechanism of hydrolysis
the
with
existence
thermolysin.
of a ternary
and amino fragment bond in place
of
an amino-enzyme lease
being
for
and other
determined
thermolysin
work. his
with
acyl
fragment peptide
an anhydride
the order
of
affinity binding
and in some cases
(5) assuming
susceptible
either
a
product
re-
of the
acyl
sites. for
or
This may
chymotrypsin
have been known as enzymes of
the
mechanism.
University We also
technical
of
respective
which
Acknowledgment. Toronto
enzyme,
via
observed
pepsin,
proposed
of the
by the relative
to their
proteases
acyl-enzyme
involvement
intermediate,
or amino moiety be true
the
cleavage
proceeds
was also
case of
is of
and This
In the
complex
after
RESEARCH COMMUNICATIONS
We wish to thank for
thank
helpful to Dr.
assistance
Professor
suggestions M. Takagi
in isotope
of
T. Hofmann of
concerning this
laboratory
this for
experiment.
REFERENCES 1) 2) 3) 4) 5)
Blow, D. M. (1976) Act. Chem. Res. 2, 145-152. Kester, W. R., and Matthews, B-W. (1977) Biochemistry 5, 2506-2516 . Wang, T. T., and Hofmann, T. (1976) Biochem. J. 153, 691-699. Newmark, A. K., and Knowles, J. R. (1975) J. Am. Chem. sot. 97, 3557-3559. James, M. N. G., HSU, I-N., and Delbaere, T. J. (1977) Nature 267, 808-813.
101
