The inactivation of pepsin by an equimolar amount of 1-diazo-4-phenylbutanone-2

BIOCHEMICAL

Vol. 26, No. 2, 1967

THE

INACTIVATION

A.

Hamilton,

IDepartment

Received

recent

catalyzed to

by

another

report

by

our

compound, the

and

Spona,

Lawrence

The Pennsylvania Pennsylvania

Park,

Rajagopalan,

of pepsin

communicate

investigate

AMOUNT

Stein,

by

results

on

and

the

State 16802”

Moore

inhibitory

/ 0

properties

of

this

-

f: -C-NH

R

CHN2

1960;

is

Knappenberger, and

in

the

which

Dawid,

*

and

reactions

transferred

Neumann,

ferases,

Berger,

1961).

In

are

diazomethyl Buchanan,

work was Princeton,

in

to another

Levin,

these

Much of this University,

prompts reactions

were

lead

because

to pepsin

f

I

transpeptidation bond

We

Cu(II)

I -CH-C-NHR’

DPB

peptide

the

ester

compound

CH2

j

on

catalyzed

(DPB).

?

catalyzes

University

(1966)

of pepsin

\

H

I-DIAZO-

Crowell

methyl

inhibition

l-diazo-4-phenylbutanone-2

possible

I cH2-

D.

diazoacetylnorleucine

/

-

OF

16, 1966

inactivation

us

Jlrgen

of Chemistry, University

December

The

PEPSIN BY AN EQUIMOLAR 4-PHENYLBUTANONE-2

OF

Gordon

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

this

known group 1963).

done New

it

is

at Frick Jersey

was

by

prepared

Chemical 08540.

portion

residue

similar

the

193

amino

1959;

inhibited

replaces DPB

acid

Katchalski,

respect be

the

amino

and

to

which

to specific

amide

(Bovey

the and

Fruton,

Fujii,

glutamine

a similar

Laboratory,

group

cleaved Yanari, and

amidotrans-

diazoketones

amino as

of

or (French,

diazomethyl

Princeton

esters

BIOCHEMICAL

Vof. 26, No. 2, 1967

analog

of

The

phenylalanine

results

amides

summarized

inactivator

DPB

the

and

Methods

and

DPB-2-C

Birkofer

(95-100°C).

1966)

and

2 or

3 times

number

Kiehs

it

synthesized

acid

(New

England

They

were

14

DPB-2-C

reacts

for

that

in

had

from

a one

DPB

pepsin. is

to one

a potent

molar

Swine

crystallized

Corp. by

activity

lyophillized)

by

the

the

swine

twice x

at

of

P2589,

from

0. 1

lo7

and

pepsinogen

obtained

and

procedure

procedure

J2211,

and were

) by

of 3.2

numbers

alcohol)

acid

distillation

synthesized (lot

dilute

crystalline,

Nuclear

a specific

pepsin

from

3 -phenylpropionic

purified

was

(1965).

K2298,

substrates

indicate

and

2-diazocyclohexanone

Stetter

specific

communication

(1947). The

dpm/mmole.

are

were

1 -Cl4 by

which

enzyme.

14

3 -phenylpropionicdescribed

this

(Crowell,

with

Material

mm

in

of pepsin

stoichiometry

(I)

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3466,

(lot

Mann

Research

Laboratories. The

pepsin

rnp

assuming

278 Enzyme

and

Chemical

Stein,

3 x

-3

10

the

However, hours

dissolved

way

as

no

the in

0. 04 M

-ca.

the

at

of pepsin acetate

10s6

M

and

was

2 by

the

ninhydrin

assay

-tyrosine

(Cycle

the the

that

C or

incubation

with

DPB pH

and

inhibited concentration

after

1. 5 to

4 hours

enzyme

which

was

no

inhibitor

38”

was

C and

pH

5.5

treated

added. for

a few

activity. DPB-2-C

5. 5 (containing

194

the

at

or

1966).

substrate

determined

except

enzyme

enzyme

initial

enzyme

the

at

(Perlmann,

to

on

buffer,

assays

absorbance

900

refers

5”

effect

pH

the

50,

inhibition

inhibited

dialysis

of

C and

these

3 x

0%

from

-phenylalanyl-L

inhibition

case

appreciable reaction

For

percent

each

extended had

For

in

38”

N -acetyl-L

substrate.

The

estimated

coefficient at

using

was

M.

was

extinction

1948) ) as

and same

solutions

determined

concentration

hydrolysis in

was

Corp.

enzyme

of

a molar

activity

(Moore

was

content

14

the

CuSO4

enzyme for

was the

reactions

Vol. 26, No. 2, 1967

catalyzed or

by

Cu(II)). 14

DPB-2-C

incubated

BIOCHEMICAL

at

A

was

added

38°C

for

solutions

which

initially

2 to

were

dialyzed

at

5 ml

least

20 hours

extinctibn as

aliquot

of

1960)

and

of For

dialyzed

the

radioactivity

and

Some

effect.

the

of the by on

characteristics

at

added

to

5°C;

buffer

rnl.r

usually

(pH

5. 5) for

dialysis

is

the

essentially

the

a 0. 1 or of Bray’s

a Parkard

was

the

at

Following

DPB

solution

incubation

acetate

278

of

the

dialyzed

15 ml

using

the the

of the

results enzyme

the

enzyme

by

DPB,

but

amounts.

by In

the

inhibition;

because

evidence

that

absence

the

diazoketone

that

of

such

at

rapidity

not

cyclohexane

extracts

of

Cu(II)

not

very

0.2

same

ml

solution

Tri-Carb

(Bray, Liquid

1.

site

Although

Cu(I1)

alone

is

which

the a slow

are

its not

at

observed;

catalyzed the

is

of phenylalanine,

195

inactivation

of

almost

in

varying

protect

the

active

site.

this

result

is

Additional

which does quantitatively

not

enzyme

the

necessary

2-diazocyclohexanone,

DPB

of

reaction.

enzyme

has

because

can effect

DPB

inactivation

present

substrate

Cu(II) on

b$

reproducible,possibly

is

of the

catalysis

there

exerts

protection

pepsin

catalyzes

Cu(I1)

DPB

that analog

Table

a specific

a specific

an

of

markedly

of

Cu(II),

observation is

is

presumably

binding is

rate

impurities

of the

inactivation

it

absence

its

of Cu(II),

expected

Although

trace

thus,

presence

in

catalysis, In the

catalyzed

inhibition

summarized

DPB.

In the

was

solution and

measurements,

determined

by

from

0. 04 M

pepsin

radioactivity

solution

were

of buffer.

inhibited

ether

Following 14

of

an

solution

time.

DPB-2-C

2 changes

the

pepsin

of

least

enzyme

is

lengths

4 liters

the

it

the

of

Discussion

shown

no

of

10 ~1)

Counter.

Results

are

10 ml

versus at

the

Scintillation

to

(ca.

contained

with

of pepsin.

aliquot

various

coefficient

that

small

AND BIOF’HYSICAL RESEARCH COMMUNICATIONS

inhibit from

for is

a the

enzyme.

aqueous

Vol. 26, No. 2, 1967

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table The

Concentration of pepsin

(M

Inhibition

1

of Pepsin

Activity

Time Incubation (minutes)

x 104) during Incubation DPB at pH 5.5, 38°C

with

Pepsin

DPB

Cu(I1)

0.3

0.83

0.0

0.3

0.83

0.0

0.3

0.83

0. 1

0.3

0.83

0.3

by

DPB

of

70 Enzyme inhibited ninhydrin at pH 2,

5

0

4oa

120

5

84

0. 1

1.20

80

0.00

1.0

120

0

0.3

0.83b

0.0

120

0

0.3

0.83b

1.0

120

76

0.3

0. oo=

0.5

120

0

however, it was usually solution also contained 1.5 c 2-diazocyclohexanone

x

a This value is not and 50. b During N-acetyl-L-phenylalanyl-L-tyrosine. M) was used instead

solution

does

results

using one

At

the

per

mole

to for

label that pepsin

14

from

which

of the

inhibited

inhibited

inhibition.

In

indicate

to

give

because

inhibitor

observed

of the

concentrations

possibly the

the

of pepsin

high

though

pepsin

reverse

DPB-2-C

decrease,

even

not

is

enzyme. enzyme

by

by

the

enzyme Table

that

one

a modified the

inhibitor

reacts

in

excess

Thioethanol

and

5. 5.

The

Vratsanos,

p-bromophenacyl

20 M x

-5 10

of

which

percent with one

mole

and The

is

itself.

result

Wasserman,

DPB

some reacts

catalytically

inhibition

hydroxylamine

thioethanol

following

summarized

molecule

only

bromide.

196

solution

2 are

pepsin

of inhibitor

present

at pH

Erlanger,

between 10m2 (6.6

DPB.

extraction

molecule

inactive. to

of

, cyclohexane

incubation

with

too reproducible; incubation the

(from assay 38°C)

appears However,

reacts do

not

is

in

Cooper,

observation

with remove contrast (1965) that

BIOCHEMICAL

Vol. 26, No. 2, 1967

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table The

Concentrations during incubation with DPB-;!-Cl4 38” c pepsin

Reaction

(M x 104) of pepsin at pH 5. 5

DPB-2-C

l4

2

of Pepsin

Time of in cuba tion (min)

with

Radio activity following dialysis (dpm/mmole of pepsin x lo-‘)

Cu(I1)

14

DPB-2-C

70 pepsin with inhibitor attached

% pepsin inhibited (from ninh ydrin assay at pH 2, 38°C)

2.3

72

86

6.5

18

7.0

120

9.3

10

1.0

30

2.6 2. 5a 2.4b

81 78a 75b

91

9.3

100

1.0

30

1.9

60

60

0.0

10

1.0

30

0. oc

5

0.5

120

4. gd

0.1

-e

3

a Following dialysis the inhibited protein solution was incubated with 0.1 M thioethanol at pH 5.5, 38°C for 24 hours and then dialyzed again for 40 hours versus 0.04 M acetate buffer, pH 5. 5. b Same as footnote ‘a’ except hydroxylamine (0. 1 M) used instead of thioethanol. c dpm/mmole of initial DPB-2-Cl*; the experiment indicates that all the inhibitor which does not react with the enzyme is removed under the dialysis conditions. d Pepsinogen used instead of pepsin. e The pepsinogen showed no catalytic activity either before of after the attempted reaction with DPB-2-C14.

pepsinogen

does

is reacting

at the active

The

inhibited

(with

trichloroacetic times

acid

with

acetone

radioactivity with

inhibitor Wasserman,

(TCA)

were

Cooper,

again

indicates

that the inhibitor

of pepsin. enzyme

can be precipitated

and the precipitated

and dioxane

to the enzyme and

the inhibitor

DPB-2-C14)

or incubation

observations

with site

is removed

6 NHCl,

(these

not react

from for made

with

no loss

the TCA

precipitated

K.

by Dr.

in tris T.

linkage

Gross

197

washed

of radioactivity.

a few hours

is by an ester 1966;

material

Fry).

and Morell,

several

1966;

the

on treatment (pH 8) at 25”

If the attachment

(Erlanger,

5 N

However,

material buffer

with

Vratsanos, Rajagopalan,

C of the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 26, No. 2, 1967

Stein,

and

Moore,

base

catalyzed

carbonyl

ester

group.

have

thiols

are

attachment

of

Fruton, be

Hishida

to

present inhibitor

increased

in

react

as

1965)

(1962)

acceleration

known

is the

must

and

rate

also

copper

and

hydrolysis

some

when

Delpierre

Newman

observed

diazoketones

of

1966;

to the

Bender

related

are

of

the

the

Silver

(1962)

systems.

However,

phenols,

(Yates,

enzyme

rate by

and

model

alcohols,

a catalyst

the

dramatically

and

with

then

amines,

1952).

The

currently

and

site

under

and

mode

further

investigation. Acknowledgement: GM-

14985

This

from

the

research

Institute

of

was

General

supported

Medical

by

GM-09585

Sciences,

and

Public

Health

Service.

References Bender, M. L and Silver, M. S., J. Am. Chem. Sot., 84, 4589 Birkofer, L., Chem. Ber., 3, 83 (1947). Bovey, F. A. and Yanari, S. S., Enzymes, 4, 63 (1960). Bray, G. A. , Anal. Biochem., 1, 279 (1960). Crowell, L. D., Senior Thesis, Department of Chemistry, Princeton, 1966. Delpierre, G. R. and Fruton, J. S , Proc. Natl. Acad. Sci. U.S., (1965). Erlanger, B. F., Vratsanos, S. M., Wasserman, N., and Cooper, J. Biol. Chem., 240, PC3447 (1965). Erlanger, B. F., Vratsanos, S. M., Wasserman, N , and Cooper, Biochem. Biophys. Res. Commun., 243 (1966). -23, French,

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C., Dawid, 2186 (1963).

J. S., U.S., E. and S. and H., 33 (1959). M. S. G.

N.,

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Buchanan,

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Fujii, S., and Knappenberger, 2, 759 (1961). Morell, J. L., Biol. Chem., Stein, W., J. Biol. Chem., Levi& Y., Berger, A., and and E.,

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