The mechanism of the activation of papain

Vol. 34, No. 5, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE MECHANISM OF THE ACTIVATION Ira Department

Received

January

8. Klein

of Biochemistry,

and Jack

OF PAPAIN

F. Kirsch

University

of California,

Berkeley

14, 1969

The predominant form of inactive papain prepared by the method of Kimmel and Smith (1) is shown to be a mixed disulfide formed between a sulfhydryl group on the enzyme and cysteine. Reduction or other nucleophilic cleavage of this bond frees the active thiol of papain and stoichiometric amounts of free cysteine or cysteine whose sulfur atom is cova {zntly bound to the activating nucleophile. It is shown specifically that K CN activation of papain produces 14C labeled 2-iminothiazolidine-4-carboxylic acid (ITC) and a stoichiometric amount of free SH on the protein. Performic acid oxidation of inactive papain yields an amount of cysteic acid approximately equivalent to the quantity of ITC obtained in the cyanide activation experiment.

Crystalline generally

papain

obtained

yet

of the

been

as thiols, inactive

Three

in Fig.

Form A has been showed molar

that

sodium

ratio

as demonstrated (7,8)

have

internal

suggested activation cially

from the

prepared

mixed

fact

were

virtually

disulfide

during

the

of papain

575

has not

of activation.

enzyme on a 1: 1

course

(6) thiol

reagents. that

of activation

and earlier is bound

activated

(9) who showed identical

The

procedure

the the

active

cyanide

by carbonyl

of Sluyterman

is

of Neumann --. et _.., al (5) who

Morihara

that

(4).

mechanisms

activated

to it

B in which

to inhibition

by experiments of papain

(Na3P03S) bound

(1)

have been made are shown dia-

the presumed

chromatography.

structure

sensitive

which

borohydride by their

by the experiments

reversibly

hemithioacetal

particularly

with

phosphorothioate

for

or sodium

suggestions

supported

by Sephadex

argued

(2,3),

and Smith

of the enzyme is promoted

of the enzyme obtained

1 together

and became

of Kimmel

Activation

form.

cyanide

form

established.

grammatically

by the method

in an inactive

by such reagents nature

prepared

papain Inactive

workers as an is form

the kinetics

to those

exhibited

and cysteine.

Glazer

C is

of

by an artifiand Smith

(4)

Vol. 34, No. 5, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Inactive

Active

-CN

\

OH

CN-

XJ I

+ CN-X w

C Figure 1. Previously suggested structures of B, (Ref. 6) ; and C, (Ref. 9)) XH = cysteine or The mode of activation of the enzyme species. Morihara (6) for structure B is shown together

did

look

formic

for acid

such a mixed oxidation

considerably

less

The results form of papain

is

disulfide

by analyzing

of the enzyme but than

the

thiol

titer

reported

herein

indeed

a mixed

inactive

titrable water

in the

thiol

per mole

to remove

sodium

phosphate

hr with

a final

absence

free buffer

(10).

Protein

that formed

activator

dialyzed

Activation

cysteic

from lots

cysteine

extensively

and the enzyme.

than

against in 0.01

was carried

by the method

out x

per-

inactive

SCA and 7DB.

and had less

was determined

576

after

were generally

that

of enzyme and K14CN of 3.6

concentration

acid

the predominant

was dissolved

The enzyme was assayed

1o-3 y respectively. IgelstrBm

enzyme.

or cystine

at pH 6.8.

concentration

of the

(Worthington

of protein)

cysteine

quantities

disulfide

of added

for

found

demonstrate

Methods rmd Materials--Papain were

inactive papain: A, (Ref. 5); unidentified inactivating by cyanide suggested by with those presumed for A and C.

Both 0.07

lots moles

distilled M EDTA-0.01

M

at 25°C for

4-5

10V4 2 and 1.0 x of Kirsch

by using

and

~~~~~ 51,000

(11).

BIOCHEMICAL

Vol. 34, No. 5, 1969

Gel filtration

was done on a 17 x 1 cm column

M ammonium acetate Thiol

a Packard

2,4

Tricarb acid

papain

buffer

with

Model

and 8 hrs

oxidized

(14).

from

cysteic

acid

using

a 25mm UM-10 membrane. Model tal

120 Amino Acid Readout

Cysteic Analyzer

2-Iminothiazolidine-4-carboxylic tography this

system

compound

was carried

column

of Bio

Rad Agl-X8

papain

was put

on the

to elute

the protein

was applied 0.09

et _al mm

(OH-),

200-400

in 1 c NH40H.

and excess

ammonia

ITC from

was separated with

Model

CRS 1OAB Digi-

(15). using

the paper

An aliquot

and isolation

of

on a 1 cm x 1 cm of K

30 ml of distilled

14

CN activated

water

a NaCl gradient

CN: ITC was eluted

chroma-

was used

(0-0.15!)

at 0.06

M - and CN- at

-M NaCl.

Results and Discussion--Fig. profile

of a 0.5 ml aliquot

Materials

and Methods.

new radioactivity with

excess

In-

a Beckman/Spinco

chromatography

and then

AIM-2,

fitted

identification

mesh.

containing

ultrafilter

was detected

exchange

8CA)

pestle.

protein

using

standards

Further

(16).

out by ion

column

to separate

Soluble

acid

of Bradham

a teflon

an Infratronics

to known

in

(lot

Manual

with

1 ml capacity

with

(13)

sodium citrate

pH 2.2

was determined

fitted

and compared

System

fitted

Diaflo acid

residue

dilutor-Instruction

by centrifugation.

an Amicon

solution

lyophillized with

homogenizer

was removed

1 ml/min.

0.4 urnmoles of papain

was extracted sample

0.01

Spectrometer.

soluble

recommended

was about

scintillation

Scintillation

The partially acid

rate

G-10 with

(12).

was done on about

a Potter-Elvehjem

material

reagent

a toluene-ethanol

oxidation

and cysteic

5) using

soluble

in

of Sephadex

The flow

Ellman’s

3003 Liquid

(Beckman/Spinco

part

as the eluent.

was counted

Performic for

pH 6.8

was titrated

Carbon-14

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

protein.

and the protein

of a papain

The last

containing The first peak.

2 illustrates

peaks (I)

solution

Peak

(III)

appear

and smaller

The middle

the

one (II)

577

one is

filtration

activated

contains which

gel

as described

unreacted

are due to the coincident

corresponds

elution

with

in

cyanide.

Two

cyanide

reaction

the

void

to a new species

volume

Vol. 34, No. 5, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

80 60 I ;

40

E” 20 0

6

15

10

20

Volume Eluted (ml) Figure 2. Separation of the products of the K 14 CN activation of papain by Sephadex G-10 chromatography as described in Materials and Methods; (0) protein, (a) carbon-14 and (A) thiol.

produced

by the reaction

material

was lyophillized

graphy

using

of the protein and subsequently

the system

of Bradham

The identification described

cally

in Fig.

under

explanation

3 where

is shown

react 14

enzyme with

with

the

is

thus thiol

sulfur

about

thiol

14

result

I,

from would

peak

chromato-

as

only

peak is

based

activating labile

cyclizes

nucleophile

must

Attack

cysteine. would

14 C while about

even if

peak

lead

of

to inactive

all

I has only

40 mumoles,

in this

the protein

9 mumoles.

although

coefficient.

was obtained

to approximately

578

species

to pro-

to 14 C-B-thiocyanoalanine.

activity that

papain

ITC (17).

on the extinction

catalytic

be equal

of

shown diagrammati-

inactive

disulfide

converted

50 mumoles

the graph

the

atom on the

cysteine

is

The latter

C than

of protein

CN- induced

evident

chromatography

with

to give

in the protein

100 mumoles

titer

to react

atom of the mixed

contains

of the maximal

of this

experiments

the sulfur

site

The titratable

It

has more

the active

Peak II

are over

cyanide

of these

preferentially

CN at the other

latter

as ITC by paper

exchange

enzyme and 6-thiocyanoalanine.

peak II

This

and Methods.

the conditions Since

identified

by ion

straightforward

duce the active

the activator.

et F”?” al (16). ..,-

was supported

in Materials

The most

with

were

0.5 mole/mole

there About

85%

experiment. active, protein.

It

Vol. 34, No. 5, 1969

therefore clusion ments

BIOCHEMICAL

follows

that

has been

drawn

(4,9,18).

not

all

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the papain

previously

molecules

by others

The unactivatable

are

activatable.

on the basis

protein

This

of different

may represent

products

con-

experiof partial

autolysis. The values cysteine titer

produced of the

performic only

shown

acid

during

analyzer

scheme given

Table

Papain

in Fig. in which

I.

Comparison acid (ITC)

Lot

acid

were

#

column

to protein

0.45,

0.51,

cannot

the inactive

of Thiol Produced

amount

exclude

papain

7DB

equal

These from

of these

to the

latter

cysteic

thiol

values acid

experiments

in mixed

are

produced

support mechanism

disulfide

of

on the

material

a more complicated

is bound

of modified

2, 4, and 8 hrs

of contaminating

linkage

the of to

Titer and 2-Iminothiazolidine-4-carboxylic by the K 14CN Activation of Papain.a

Thiol

ITC

p;FOlr8&

8CA

amounts

for

and 0.51.

the results

3, they

the

ratios

separation

of a small

Although

that

are approximately

due to the incomplete

the oxidation.

activation

cleavage

Cysteic

oxidation

qualitative

amino

I demonstrate

by cyanide

enzyme.

acid

in Table

P;FOK%

%

%

40.5

52.5

43.3

56.2

56.0

a.Based on protein concentration the maximum obtainable activity.

61.0

as determined

579

from

absorbance

at 280 mu and

Vol. 34, No. 5, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

N III, /NH: s-s
ic

coo-

CZN-

SH + S,

-

CH2-C(-l cool H H ‘N’ *p -QNH s’ \

Figure

labile

cysteine

forms

the

through

active

of external

cysteine

to form the

dithio

bridges

remains

a thiol

site.

change

it

Reaction

3.

of cyanide

group

Activation as shown

active

in the

different

would

in Fig. It

site.

or not

this

papain.

that

proceed

3 followed has been

inactive

from

then

enzyme can be broken

to be seen whether

with

I ,CH CH2 lcoo-

which

eventually

in two steps;

by an internal

shown that

without

loss

property

is

release

disulfide

one of the

internal

of activity

(19))

related

ex-

but

to the activation

process. The mixed

disulfide

streptococcal

protease,

is

with

associated

disulfide Further

formed

in which

the inactive

binding

related

papain

a volatile form

from human plasma

experiments

and the apparent

form of inactive

but

as yet

of the enzyme

mercaptalbumin to the reaction

of activators

is analogous

will

cation.

580

with

unidentified

(ZO),

of papain

of a

mercaptan

and with

and cysteine

be reported

that

the mixed

(21).

with

in a later

carbonyl

reagents

communi-

Vol. 34, No. 5, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGVENTS

We are grateful helpful

to Drs. J. Drenth, J. N. Jansonius, and R. Koekoek for

discussion of this problem.

This research was supported by National Institutes

of Health, Grant

GM12278and National Science Foundation Grant GB4606.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21.

Kimmel, J. R., and Smith, E. L., J. BioZ. Chem. 207, 515 (1954). Chem. 238, 165 (1963). Sanner, T., and Pihl, A., J. Biot. Smith, E. L. and Kimmel, J. R., in Boyer, P. D., Lardy, H., and Myrbgck, K The Enzymes, Vol. 4, Academic Press, New York, 2nd ed., p. 133 (260). Glazer, A. N., and Smith, E. L., J. BioZ. Chem. 240, 201 (1965). Neumann,H., Shinitsky, M., and Smith, R. A,, Biochem. 6, 1421 (1967). Morihara, K., CT. Biochem. (Tokyo) 62, 250 (1967). Bergmann, M., and Ross, W. F., J. BioZ. &em. 111, 659 (1935). Bergmann, M., and Ross, W. F., J. Biol. Chem. 114, 717 (1936). Sluyterman, L. A. AE., Biochem. Biophys. Acta 139, 430 (1967). Kirsch, J. F., and Igelstrllm, M., Biochem. 5, 783 (1966). Glazer, A. N., and Smith, E. L., J. BioZ. C&em. 236, 2948 (1961). Ellman, G. L., Arch. Biochem. Biophys. 82, 70 (1959). Hall, T. C., and Cocking, E. C., Biochem. J. 96, 626 (1965). Hirs, C. H. W., J. Biol. Chem. 219, 611 (1956). Hirs, C. H, W., Moore, S., and Stein, W. H., J. BioZ. Chem. 235, 633 (1960). Bradham, L. S., Catsimpoolas, N., and Wood, J. L., Anal. Biochem. 11, 230 (1965). SchUber, I. A., Kawohl, M., and Hamm,R., Ber. 84, 571 (1951). Bender, M. L., Beg&-Cant&, M. L., Blakely, R. L., Brubacher, L. J., Feder, J., Gunter, C. R., Kkzdy, F. J., Killheffer, J. V., Marshall, T. H., Miller, C. G., Roeske, R. W., and Stoops, J. K., J. Am. Chem. Sot., 88 5890 (1966). Neumann,H., IsmeZ J. C%m. 5 87~. (1967). Ferdinand, W., Stein, W. H., "andMoore, S., J. Biol. Chem. 240, 1150 (1965). King, T. P., J. BioZ. Chem. 236, PCS (1961).

581