A reactive aspartyl residue of pepsin

BIOCHEMICAL

Vol. 30, No. 5, 1968

A

REACTIVE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ASPARTYL

RESIDUE

OF

PEPSIN

:x Keelin Charles

Oh-Kil

of

Fry

F. Kettering Research Yellow Springs, Ohio

Kim,

Department

T.

J&-gen

Spona,

Chemistry,

and

The

University

Laboratory 45387

Gordon

A.

Pennsylvania

Park,

State

Pennsylvania

*;I;

Hamilton University

16802

Received January 26, 1968

Recently

it

was

inactivated

The

results

pepsin.

the

the

the

pepsin

group

be

present

of an

specifically

, refluxing

by

the

active

site that

that

the

amino

Be-Val-Asp-Thr.

another

class

a different

of DPB

(HPB) acid

This of

pepsin

section

of

the

aspartyl

inhibitors,

Cooper,

1966). pepsin

molecule

catalysis.

the

materials

Spona,

method

of McPhee

obtained

from

the

crude

-2

NaOH

in

methanol

with for

2 hours,

and

and

methods

Crowell, and

1967).

Klingsberg

product

evaporation

HPB

most

was A

reaction

acidification of

the

(1944).

of the

by:

were

of the

of to

pH

solvent,

* *,~ Contribution Alfred P.

(DPB).

indicating

Wassermann,and

enzymic

noted, (Hamilton,

of the was

near

and

is:

attacked

the

or

evidence

residue,

Vratsanos,

in

-2

l-hydroxy-4-phenylbutanone-2

aspartyl

define

pepsin

Methods

previously extension

of

that

-4-phenylbutanone at

report

1967)

Crowell,

1 -diazo

residue

results

involved

we

ester

(Erlanger,

phenylbutanone water

an

that

and

material

form

from

Unless

an

to

and

occurred

communication

different

the

of

reaction

aspartyl

may

given

the

this

Therefore,

Spona,

amount

containing is

Materials

equimolar

present

a-haloketones

which

(Hamilton,

that

S-carboxyl

sequence residue

an

indicated

with

with

reported

by

In

reacts

was

No. 294 of the C. F. Sloan Research Fellow,

Kettering 1967-69.

489

Research

Laboratory.

same prepared

as

those by

crystalline 1-chloro-45,

addition extraction

of with

BIOCHEMICAL

Vol. 30, No. 5, 1968

ether,

distillation

product H,

at 112-114’C/2

(analysis:

7.43)

talc.

melts

pected

The

described

(AcHPB)

anhydride.

evaporation

AcHPB

spectra H,

was

prepared

7.37:

The

by refluxing

was

for

H,

1944).

of the solvent,

(talc.

from

ether.

found;

The

C, 73.41;

nmr, and mass spectra are those exmethanol (1 max , 274 rnp, E, 19,300) of HPB

and Klingsberg,

(m. p. 43.5-44.5”(Z)

and analysis

the solid

identified

C12H1403;

ester,

l-acetoxy-4-

HPB

with

was

recrystallized

by its IR,

excess

nmr,

acetic from

and mass

C,

69. 88; H,

6. 84; found;

were

prepared

by reacting

C, 70.02;

6. 85). Large

swine

quantities

pepsin

an acetate

for

(0.05

radioactivity

at 4°C

-ca.

times

pepsin

Mann

M,

The

were

in the dialysate) by adding

the precipitated

material

85% alcohol,

of Cu(I1)

dialyzed versus

and dried

the acetate

labeled.

The

dissolves

with

Dansyl-Ile dansyl

amino

acids

obtained

Woods

labeled difficulty was

on a Phoenix Results

readily

were

after

were

purchased

from

no further

After

sitting

in vacua.

The

overall

-

washed

was

was labeled

88% of the pepsin

in solutions

14

over-

recovery

60% of the pepsin used,

at 38°C

and the C

by centrifugation,

amino

of Morse Pierce

of the peptides

by thin-layer

performed

automatic

buffer,

separated

dissolves

by the method

degradation

(1967)

M

was

of pH 6 or above

and

in pH 5 solution.

prepared

acids

and Wang analyses

pepsin

(until

of alcohol.

75%; when 5 x 10m4M DPB-2-C14 was used, 14 14 C , and when 10 x 10m4M DPB-2-C was

with

(1 x 10m4M)

exhaustively

10 volumes was

-4

Lot no. R3456) in -4 l4 (5 or 10x 10 M; specific

DPB-2-C

then

5 x 10

Laboratories,

in the presence

precipitated

with

Research

pH 5. 5) with

solutions

appeared

pepsin

several

-labeled

x IO8 dpm/mmole)

2 to 2. 5 hr.

night

14

(2x crystallized,

1.62

labeled

of C

buffer

activity,

acid

C, 73.15;

phenylhydrazone

phenylbutanone-2

ether.

and recrystallization

and its IR,

(McPhee

After

mm,

C10H1202;

at 44-45”C,

for HPB.

has been

for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Chemical were

chromatography

by the method acid

and Horecker Co.

(1966).

Other

dansyl

amino

as described

by

The

identified

on polyamide

of Spackman,

sheets.

Stein,

Amino

and Moore

(1958)

analyzer.

and Discussion

At slightly released the reaction

from

basic

pH’s, a radioactive, 14 -labeled enzyme. the C

follows

first

This result -ca. 5 hours. attached at one specific

order suggests site.

ether-extractable

compound

In 0.05

M tris

kinetics

to over

80% completion

that

essentially

Preliminary

pH 8. 05, at 3O”C, and tl,2

all the radioactivity

chromatographic

490

buffer,

is rapidly

experiments

is is sug-

BIOCHEMICAL

Vol. 30, No. 5, 1968

gested

that

the ether-extractable

shown

in Table

1 verify

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

radioactive

this

conclusion.

Equivalence

of HPB

is HPB.

prepared

and the Radioactive

Compound

Released

Total Radioa_cgtivity (dpmx 10 )

-2 x 10 mmole (88%) pepsin

1.46

(2)

ether-extractable after hydrolysis

labeled

the

at pH 8

1620b

17.7 pepsin

(3)

phenylhydrazone plus 1.86

prepared from mmoles HPB

(4)

recrystallized (3)

phenylhydrazone

from

2.25

8.6

(5)

recrystallized (4)

phenylhydrazone

from

0.29

8.2

(6)

expectedC specific phenylhydrazone

activity

from

Specific Activity (dpm/mmole x 10 -“I

20.8

radioactivity of labeled

results

1

Descriptiona

(1)

The

The phenylhydrazone

Table The

compound

(2)

11.8

8.5

of the

9.4

a The labeled pepsin (1) was incubated in 45 ml 0. 05 M tris buffer, pH 8.05, for 48 hours at room temperature. The solution was extracted several times with ether, the organic layer was dried and evaporated, and the residue dissolved in ethanol. The ethanol solution after filtration was counted (2). To the ethanol solution was added 1.86 mmoles unlabeled HPB, and the phenylhydrazone of HPB was prepared by adding phenylhydrazine and a few drops of 1 N HCl. The solid phenylhydrazone (3) was recrystallized twice from ethanol to give (4) and (5). b Specific activity of the DPB-2-C l4 used to prepared the labeled pepsin. c Calculated from the specific activity of the DPB-2-C14, the known amount of unlabeled HPB added, and the total radioactivity in (2) assuming it is all due to labeled HPB.

radioactive

compound

repeated

is only

extractable The

Since

slightly

radioactive rate

(AcHPB), stinilar

8.5

higher compound

of hydrolysis

was

hydrolysis range

unlabeled

recrystallizations.

hydrazone

rate

plus

to 9.6,

was

at 3O”C,

than

that

not change

must

specific

observed,

its

specific

activity very

nearly

activity

on

of the phenylall

of the ether-

be HPB. compound,

to see if simple

observed

of AcHPB

does

the expected

of the model

investigated to that

HPB

for

release

followed

using

esters of HPB

of HPB from

a Radiometer

in 5% dioxane-water,

491

I-acetoxy-4-phenylbutanone-2 would

the labeled pH stat.

the second

order

hydrolyze pepsin. Over

at a The

the pH

rate

constant

BIOCHEMICAL

Vol. 30, No. 5, 1968

for

the base

catalyzed for

PH 8, $2

for

of AcHPB

the release

structure

group

For

of AcHPB

the hydrolysis

observed *e tl/2 that a reasonable carboxyl

hydrolysis

for

of labeled

digested

with

native

pepsin

(60% labeled)

was

dissolved

a slightly

(0. 1 M,

turbid

was

pepsin

essentially

all

brought

to pH 2.02,

went

of pepsin

solution

of 21 hr.

The

was

digest

5.0 to neutralize

was

283-292

G-15

a C

paper

(69,

electrophoresis

a

a single -labeled

regions

labeled

tetrapeptide,

was

in 0.2

(b) paper

in 8% formic

1959).

(Figure

(a)-(c)

respectively)

of the chromatograms.

The after

69 hr.

492

buffer,

acid.

1). The

in the original

and lyophilized

stage

by performing

Gel of From in succession

acid-water,

5 hours

hydrolysis

flat

at 40 watts/cm;

acid-water,

of purification

acid

200:30:75,

on a Savant

of radioactivity amino

of

digest.

780/O recovery.

electrophoresis

was

recovery

to dryness.

with

for

pH

After

on a 0.9 x 157 cm column

acid

ml

a total

the digest

in pyridine-acetic

At each

2.35 for

M acetic

precipitate,

isolated

was

(pH 2. 95) was

in l-butanol-acetic

at pH 3.6

determined

to continue

solution,

acid

In a few

an additional

ml of 1. 0 M acetate

bf white

solution

the solution

0.2

of radioactivity

1966);

apparatus

when

in

of the

ml of a pepsin

hours,

suspended

tetrapeptide

Digestion

allowed

pooled

buffer

by adding

was

present

and Anfinsen,

narrow

4.3

90% of that

chromatography

91% in steps

After

G-25

peak

temperature

ml in 0. 01 M HCl).

resulting

M acetic

2.35

of inhibited

0.3 ml tris

M HCl.

had formed

amount

I) were

electrophoresis

Dreyer, 96, and

with

pH 7. 2) to

to 2.02

which

with

The

per

of sephadex

in 0.2

and Margoliash,

and (c) paper (Katz,

14

was (peak

Sephadex

plate

HCl.

a small

222-355

of the residue

(Nolan

treated

on a column

gave

pepsin

and the residue

filtration

v/v

then

to remove

(a) descending

to

C 14-labeled

at room

adjusted

by adding

and digestion

the excess

chromatographed c14 .m fractions

initiated

solution,

added

to dryness

material

of HPB

(0. 05 M,

standing

and 0. 1 ml of 1.0

the precipitate, into

After

the pH was

M HCl was

buffer

to pH 7. 8 by adding

Worthington

minutes

this

at

and indicates

A 133 mg sample

in 2. 5 ml tris

10 min,

at 37°C

(5 mg of 2x crystallized

Fractions

enzyme

denatured

as follows.

brought

-ca. ml of 0.02

16.5

centrifugation

. Thus,

is comparable

is an ester

fragments,

of pH 6.0.

pH 8. 5). After

lyophilized

the labeled

pepsin

peptide pepsin

solution

the solution

succession 14 C -labeled

from

This

-1

set

of pepsin.

the preparation

30 min,

is 2 to 3 hours.

the labeled

was

for

-1

is 56-+ 7 1 -mole

of HPB

pepsin

yield

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1:10:289 good

was

composition

recovery

obtained of the

in constant-boiling

in

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 30, No. 5, 1968

1.6

-

1.4

-

.6 $ z .4 I 8 .2 2 4

FRACTION

NUMBER

Figure 1. Gel filtration at room temperature of the pepsin digest of denatured, Cl*-labeled pepsin. The 1.9 x 243 cm column, which was packed with Sephadex G-25 (fine bead form) in 0.2 M acetic acid (Eaker, 1962)) had a void volume of 27 5 ml. The digest sample volume was 3.7 ml and the eluant was 0.2 M acetic acid. Two-ml fractions were collected at a flow rate of 28. 5 ml/hr. The ninhydrin color (absorbancy at 570 ml) of base-hydrolyzed 0.05 nil aliquots of each fraction was determined by the method of Rosen (1957).

HCl

at llO”C,

acids

were

peptide

is: present

group

of the Asp

the aqueous

under

comparable

amino

N-terminal

amino 1967).

dation. into

with

acid

Most

the organic

compound because

at 37”C, ether.

with

labeled

of this

liberated

at each

solution

is 1.0.

result

and free

stage

is similar

shown

is incubated

way as to pepsin.

and identifying

2 steps

493

does

to be Ile-Val-Asp-Thr

the first

the

technique

the labeled

the radioactivity found

the f3-

obtained 14 that the C -

by the dansylation

was

tetra-

to that

and suggests

with

threonine

amino

can be extracted

associated

step,

Also,

tetrapeptide

in the same was

Other

tetrapeptide

procedure,

during

the third

0.95.

In the labeled

the labeled

pepsin,

peptide

of the radioactivity

solvent,

of Asp. to Asp

to the tetrapeptide

degradation

following

Val,

the radioactivity

This

Edman

in the aqueous However,

4 hr.

sequence

the subtractive

0.89;

at pH 6. 5. If the labeled

is attached

acid

Thr,

5% of the amount

is not free

conditions

compound

0.95;

of radioactive

solution

by using

remained

than

pH 8. 5, for

from

(Gray,

Ile,

on electrophoresis

buffer,

The

to less ratio

not migrate

labeled

(1.00);

the molar

carboxyl

in tris

Asp,

peptide

of the Edman was

degra-

extracted

in the aqueous

layer.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 30, No. 5, 1968

These

results

carboxyl

group

The with

indicate

two

of the

similar acid

II indicate

it

liminary

in

materials results

II

the

will

be

further

and

III

a labeled

(Figure

for

is

attached

to

of an

sequence

peak

Ile-Val-Asp.

necessary

to that

the

p-

III

of

purified

of the

labeled of

Val,

indicate work

these

a single

by

Asp,

from

and

that

it

with

larger

pro-

tetrapeptide.

material

conclusions.

aspartyl

associated

and

sample

Further

substantiate

only

impure

composed

from

to be

separated

purification

tripeptide

material

1) appears

been

the

analysis

suggest

compound

have

employed

on

with

-labeled

which

radioactivity

contains

14

residue.

peaks

those

experiments

tripeptide

C

peptides

to

and

the

aspartyl

radioactivity 14 C -labeled

cedures Amino

that

Thr.

contains

Prea labeled

amounts

However,

residue

on

peak

pepsin

of these

reacts

with

DPB. All

of the

reacts

with

specific will

be

in

will

the

also

be

of

ester

which

is

catalysis, not

interest

present

same

see

if

Stein 1967;

or

in Moore,

to

different

are

consistent

of HPB in

the

whether

(Dopheide,

Perlmann,

at the

an

determine

(Rajagopalan,

and

occur

is

here

form

ensymic

pepsin

compounds Ong

to

tetrapeptide of

to residue

necessary

section

reported

pepsin

aspartyl

involved This

results

the and

Erlanger,

with

with

the

sequence this

in

the

known

sequence

observed

Vratsanos,

that

group

of

the

1967;

of

this the

Delpierre

Wassermann,

one work

intimately sequence

carboxyl

Stepanov

reactions

1966;

is

enzyme

DPB

Further

residue

where

Moore,

(3-carboxyl

aspartyl

Stein,

hypothesis

Ile-Val-Asp-Thr.

and

and

the

occurs. terminal

et al,

1967).

of pepsin

with

other

and

Fruton, and

It diazo

1966;

Cooper,

1967)

sites.

Acknowledgements We are indebted to Mr. David Rifenberick excellent technical assistance. This research grant GM-14985 from the Institute of General Service.

and Mr. Eugene Loewer was partially supported Medical Sciences, Public

for by research Health

References Proc. Natl. Acad. Sci., U.S. 2_6,1817. Delpierre, G. R., and Fruton, ? S. (1966) ind Stein, W.H. (1967) J. Biol. Chem. ,=,1833. Dopheide, T. A .p. I Moore, S., The Rockefeller Institute, p. 29. Eaker, D. L. (1962) Ph.D. Dissertation, Erlanger, B. F., Vratsanos, S. M., Wassermann, N. and Cooper, A.G. (1966) Biochem. Biophys. Res. Commun. 23, 243. Erlanger, B.F., Vratsanos, S.M., Wassermann, N. and Cooper, A.G. (1967) Biochem. Biophys. Res. Commun. 28, 203. Gray, W.R. (1967) Methods in Enzymology, 11, 469.

Vol. 30, No. 5, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Hamilton, G. A. , Spona, J. and Crowell, L.D. (1967) Biochem. Biophys. Res. Commun. 26, 193. Katz, A.M., Dreyer, W. J., and Anfinsen, C.B. (1959) J. Biol. Chem., =,2897 E. (1944) J. Am. Chem. Sot., 66, 1132. McPhee, W. D. and Klingsberg, Morse, D. andHorecker, B.L. (1966) Anal. Biochem., l& 429. Nolan, C. and Margoliash, E. (1966) J. Biol. Chem. 241, 1049. Ong, E. B. and Perlmann, G. E. (1967) Nature, 215, 1492. W.H. and Moore, S.566) J. Biol. Chem., 3, 4295. Rajagopalan, T. G., Stein, Rosen, H. (1957) Arch. Biochem. Biophys. 67, 10. Spackman, D. H. , Stein, W.H. and Moore, S. (1958) Anal. Chem., 30, 1190. Stepanov, V. M., Ostoslavskaya, V.I., Krivtzov, V. F., Muratova, G. L. and Levin, E.D. (1967) Biochem. Biophys. Acta., 140, 182. Woods, K. R. and Wang, K. T. (1967) Biochem. BGhys. Acta, 2, 369.

496