Vol. 38, No. 1, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MODIFICATION OF CARBOXYL GROUPS IN THE ACTIVE SITE OF TRYPSIIW A. Eyl+ and T. Inagami Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37203
Received
November
5, 1969
Summary - The chemical modification of carboxyl groups in chromatographically homogeneous bovine 5-trypsin using the carbodiimide nucleophile procedure resulted in complete loss of enzyme activity and the modification of 8 carboxyl groups. Under similar reaction conditions, the presence of a competitive inhibitor was found to decrease both the extent of modification and the loss of activity, giving a product with 7 carboxyls modified and 30% of the original activity. The carboxyl group protected to the largest extent was identified as Asp-177, with Asp-182 being protected to a lesser extent. Direct evidence is also presented indicating that residue 177 is indeed aspartic acid rather than asparagine. Specific of lysine
action
and
arginine
of substrate. strongly
suggested site.
modification
The
presence
By
identify
those
reports
the
using from
groups
suggested
an anionic
site
of competitive
a nucleophile of the
competitive
residues
by using
identification
of the
enzyme
supported Fellow.
one
proteins
by NM
is also
grant
149
for
Koshland
in the the
binding carboxyls
possible
label. thus
method
in the the
(3) led
to protect
activity
group
and
inhibitors
a radioactive
binding
of a water-
present
be
the
a combination
carboxyls
should
groups
(1, 2) also
specific with
carboxyls
for
carboxyl
by Hoare
it
carboxyl
inhibition
of a mild, in
modification,
This work was NASA Predoctoral
the
of at least
the
on the
involving
development
and
identification
site
* t
the
carbodiimide
binding
modification
long
on linkages
studies
of carboxyl
to attempt trypsin.
has
Systematic
specificity
soluble
of trypsin
labeled.
described.
GM-14703.
site
us of
in
to subsequently This
communication Effect
of the
the
Vol. 38, No. 1, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS Chromatographically cial
bovine
Shaw was
performed
.
was
performed
the
Details
from
in
essentially
method
of the
of Edman
using
with
column
tris by amino
pH-stat
using
I-14C-glycine as
Begg
(7).
buffers, Protein
acid
analyses
and
using
p-nitrophenyl
p’-guanidino
RESULTS The resulted fication phenolic
reaction in
of P-trypsin
complete
loss
of 8 carboxyl groups
activity
was
reacted
tyrosines
as well
entirely
due could
AND with
(Fig.
was
(Beckman
(Whatman
activity substrate,
bensoate
by
accomtype
15A)
DE -32) were
was
out
deter-
assayed
by a
or by active (9).
DISCUSSION EDC
of enzyme
groups
50-x8
as
(6)
carried
concentrations
ester
was
--et al.
of peptides
Enzymatic ethyl
degradation
Blombick
-cellulose
as
1 -14C-Glycin-
1.
PTH-derivative
peptide
(8).
benzoyl-L-arginine
titration
by
Purification
DEAE
(3) using
Edman
on Dowex
and
Koshland
of glycinamide
(5).
to the
and reaction
to Fig.
outlined
commer-
modification
and
presence
legend
chromatography
buffer.
mined
site
and
pyridine-acetate
employing
in the
thiazolinone
The
by Hoare in the
from
of Schroeder
studies.
EDC* given
obtained
procedure
as described
synthesized
conversion
plished
modification
are
3 stages
P-trypsin
by the
carbodiimide
nucle ophile
with
in all
essentially
a water-soluble
amide
(Worthington)
used
METHODS
homogeneous
trypsin
(4) was
AND
in the
activity 1).
as carboxyl
EDC
groups
to modification be regenerated
presence
within is
3 hours, known
(10).
and
to modify
However
of carboxyl without
of glycinamide
groups
significantly
, the
tyrosine loss
since affecting
* Abbreviations: EDC, 1 -ethyl-3 -dimethylaminopropyl carbodiimide; PTH, phenylthiohydantoin; AECys, S-(/3-aminoethyl)-L-cysteine; DFP, diisopropyl fluorophosphate. 150
modi-
the
of
Vol. 38, No. 1,197O
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IO ,-“A,,,,,,,,-,,,,,,,-..
8
6 4 2 0 0
25
50
75
100 125 150 175
TIME (MINUTES) Fig. 1. presence
Modification and absence
of trypsin with of benzamidine.
EDC
and
glycinamide
in the
p-Trypsin (10 mg/ml) and l-l4 C-glycinamide (1. 0 M) were dissolved at pH 3 in water, the pH readjusted to 4. 75, and the reaction initiated by addition of EDC to a concentration of 0. 1 M. Identical quantities of EDC were added at 1 and 2 hrs. pH was maintained at 4. 75 with 0.5N HCl on a pH-stat at 25°C. Aliquots were removed at intervals, quenched in 2 M sodium-acetate pH 3.5, and dialyzed exhaustively against 0.7 mM HCl at 4°C. Radioactivity incorporated was determined by scintillation counting with the aid of a thixotropic gel, and enzyme activity of the aliquots assayed using benzoyl-L-arginine ethyl ester as substrate. Benzamidine. HCl when present was at a concentration of 0.4 M. Otherwise, 0.4M KC1 was added to maintain ionic strength. ( A --- A ) Incorporation in absence of benzamidine ( o --o ) Enzyme activity in absence of benzamidine A ) Incorporation in presence of benzamidine (A Enzyme activity in presence of benzamidine (.-.I
enzyme
activity
complete ficity
inactivation site
resulted in
determined
the
presence
rate
of activity
10s s.
may
and
extent
About
30%
7 carboxyls
had
Labeling
of the
been
of the
modified
residues
initial (Fig.
protected
151
of the
reaction
of trypsin,
modification activity
ob se rved speci-
of chymotrypsin
modification
inhibitor of the
The
(9).
modification
treatment
The
competitive
titration
reflect
inactivation.
of the
the
site
as similar
group(s), 60%
decreased
and
of trypsin
carboxyl in only
by active
(11,12)
carried
out
benzamidine,
as well
as the
remained
after
extent 3 hours,
1). by the
inhibitor
was
achieved
by
Vol. 38, No. 1, 1970
reaction
carried
hours
with
EDC
benzamidine. the
amide
for
p-Trypsin
removal
of the
protein absence
1. 1 residues
of labeled
was
8 M
urea
for
tryptic
glycinamide AEcys
had
1.
from
labeled
glycine
peptide
corresponding
most with the
the
acid.
degradation. chromatography
in the
aspartic residues acid
The
of Dowex
reacted
carboxyl
and
unlabeled
was
for
this
was
(16)
the
also
from
Worthington
50 break-through 152
in
with
and
attached site
the
contained
to Asp-177 of labeling.
(14, 15) showed
to obtain
isolated
as
The se findings
predominant
the
Glyl,
peptide
found
is
trypsinogen
obtained
AspI,
A radioactive
of peptide.
purpose
of 14C -
sequence,
glycinamide
desirable
For
was
to S-amino
peptide
moles
of Asp-177.
mole
being
subjected
the
trypsinogen
radioactive
177-192
peptide
of trypsin.
composition:
identifies
180-189 per
seemed
oxidized
mole
radioactive 0.61
acid
p-carboxyl
sequence
is
in the
EDC
was
major
uniquely
former
proposed
with
contained
to residues
of the
more
by
1-14C-glycin-
resulted per
obtained
amino
to the
it
performic
The
177-179
177 as asparagine,
comprising
(13).
following
attached
Asp-182,
actually
thus
composition
residues
and
to partially
once
protein
of 14C-glycinamide
that
Since
the
The
arising
moles
due
50 chromatography
and
This
M
reagents
EDC
3
1 hr.
digestion
by Dowex
with
for
of 0.4
excess
glycinamide
treated
radioactive-labeled and
incubated
treated
presence
and
of inhibitor.
protein
first
in the
inhibitor
was
labeled in
was
glycinamide
heterogeneity
ethylation
indicate
stages.
peptide
The
Serl,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
possible
glycinamide
and
unlabeled
in the
of
the
0.34
and
3 hours
avoid
obtained
in two
lyophilized
incorporation
sites,
out
After
dialysis,
To
BIOCHEMICAL
direct
evidence
“active
serine”
a tryptic
trypsin
and
pure
form
fractions.
residue
digest
subjected
that peptide of to Edman
by DEAE-cellulose The
disappearance
it
Vol. 38, No. 1,197O
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1. Amino acid composition of “active from a tryptic digest of oxidized Worthington one Edman de gradation. Amino Acid
Before Edman (residues) 1.00 1.87 2.44 0.97 0.98 3.81 1.29 2.02
LYS Asp Ser Glu Pro GUY Val Cysteic
serine” trypsin
After Edman (residues) 0.85 1.04 2.67 1.00 1.05 4. 00 1.41 1.93
(l)* (2) (3) (1) (1) (4) (2) (2)
* Numbers in parenthesis indicate expected on the sequence of residues 177-192 (13).
of aspartic
acid
obtained
using
of the
N-terminal
(Fig. the
2).
after other
conditions
were
of only
20-25s
obtained
with
chromatographically 177
did
not
that
residue The
from
determines
some
arise
finding
that
that
residue
specificity
that
ionic The
of trypsin.
and
deamidation
of residue
We must
carboxyl
binding recent
residue inhibitor
conclude
protected strongly
site
which
discovery
* We recently became aware via a note added to the paper that both Hartley and Walsh have independently arrived 153
Similar
*
major
the
to (7) but
3H-DFP-treated
of a competitive
provides
thiazolinone
laboratory.
procedure.
acid.
is the
binding
our
indicating
asparatic
Asp-177
by the this
the
indeed
of the
from
in either
chromatography
of PTH-asparagine in
isolated
trypsins,
as an artifact
177 is
deamidation
peptides
results
of PTH-asparagine
conversion
as determined
purified
modification
suggests
in
based
thin-layer
no trace
for
ratios
1) confirms
However,
gave
employed
result
(Table
(17, 18).
PTH-derivative
The
to an extent
degradation
methods
PTH-derivative
results
Edman
peptide obtained before and after
largely by Smith
of Smith and Shaw (18) at the same conclusion
Vol. 38, No. 1, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a.
b.
0 0
0 0
Q PTH-. Sdrnple 0 PTH-
P;HSer
Asp
Asn
0
PTA, Asp
I
PTL Asn
Saiple
Thin-layer chromatography of PTH derivative obtained Fig. 2. the N-terminal residue of “active serine” peptide isolated from Worthington trypsin. a. Silica gel G with chloroform/methanol, Silica gel G with chloroform/formic acid, 100:5, as solvent. b. N -te solvent. Spots were localized with an Iodine-azide spray. PTH-derivative was spotted at position marked “sample”.
and
Shaw
(19)
that
an autolytic
split
reduces
affinity
of the
enzyme
further
support
to this
conclusion.
in
a peptide
sequence
adjacent
to the
residue
(Ser-183).
by
2 amino
as
shown
homologous
acid below
which
at residues
for
is
cationic
not
homologous
sequence
than
substrates
Interestingly
which
Furthermore, residues
176-177
this the
drastically and
enough,
inhibitors
the
non-homologous
corresponding
occurs
but
active
is
seryl
sequence structure
lends
Asp -177
to chymotrypsin contains
from oxidized 9:1, as rminal
is
longer
of chymotrypsin
(14, 15).
Trypsin
170 177 -GLY-tyr-leu-glu-gly-gly-lys-asp-SER-CYS-gln-GLY-ASP-
Chymotrypsin
-GLY
-ala-ser
-gly-val-ser
-
182
SER-CYS-met-GLY-ASP-
Acknowledgment - The authors wish to express sincere thanks to Mr. J. D. Ford and Miss L. H. Clack for amino acid analyses and to Mss. A. Knapp and Mr. R. Packer for able technical assistance.
154
Vol. 38, No. 1,197O
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Inagami, T., J. Biol. Chem., 239, 787 (1964). Mares-Guia, M. and Shaw, E., J. Biol. Chem., 240, 1579 (1965). Hoare, D.G. and Koshland, D.E., Jr., J. Biol. Chem , -’242 2447 (1967). Schroeder, D.D. and Shaw, E. J., J. Biol. Chem., 243, 2943 (1968). Anderson, G. W. , Zimmerman, J. E. , and Callahan, FM. , J. Am. Chem. Sot., 86, 1839 (1964). BlombLck, B., Bxmback, M., Edman, P , and Hessel, B., Biochim. Biophys. Acta., 115, 371 (1966). Edman, P., and Begg, G., Eur. J. Biochem., 1, 80 (1967). Spa&man, D.H., Stein, W.H., and Moore, S., Anal. Chem., 30, 1190 (1958). Chase, T., Jr., and Shaw, E., Biochem. Biophys. Res. Comm. 29, 508 (1967). Carraway, K. L. and Koshland, D.E., Jr., Biochim. Biophys. Acta., 160, 274 (1968). CaGway, K. L., Spoerl, P., and Koshland, D.E., Jr., J. Mol. Biol. , 42, 133 (1969). Abita, J. p. and Lazdunski, M., Biochem. Biophys. Res. Comm. 35, 707 (1969). Raftery, M.A. and Cole, D.R., Biochem. Biophys. Res. Corm-n., 10, 465 (1963). Walsh, K.A. and Neurath, H., Proc. Nat. Acad. Sci., 52, 884 (1964) Mikes, 0.) Holeysovsky, V., Tomasek, V., and Sorm, F., Biochem. Biophys. Res Comm., 24, 346 (1966). Hirs, C.H. W., Meth. Enzymol. XI, 197 (1967). Dixon, G.H., Kauffman, D. L., and Neurath, H., J. Biol. Chem., 233, 1373 (1958). Holeysovsky, V., Alexijev, B. , Tomasek, V. , Mikes, O., and Sorm, F., Coll. Czech. Chem. Corm-n., 27, 2662 (1962). Smith, R. L. and Shaw, E. , J. Biol. Chem. 244, 4707 (1969).
155