Modification of carboxyl groups in the active site of trypsin

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

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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).

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