Comparison of α-chymotrypsin and subtilisin BPN1: Size and specificity of the active site

Vol.

35, No.

2, 1969

BlOCHEMlCAL

COMPARISON’

OF SIZE

Shionogi

Research

BlOPHYSlCAL

a-CHYMOTRYPSI

AND

Kazuyuki

AND

N

SPECIFICITY

Morihara,

Shionogi

AND

COMMUNICATIONS

SUBTILISI

OF

THE

ACTIVE

Oka,

and

Hiroshige

& Co.,

Ltd.,

Tatsushi

Laboratory,

RESEARCH

N

BPN’:

SITE Tsuzuki

Fukushima-ku,

Osaka,

Japan

Received March 17, 1969 It is a well-known

fact

enzymes

a-chymotrypsin

example,

an active

center

histidine

has been

clarified

a high

as follows.

by ZPCK

but

and

a suitable tilisin

reacts

the

enzymes

(Show

acid

residues

amino

Tsuzuki, BPN’

(an alkaline

1969). was

with

proteinase

DFP*

and

and

shown

by Show

and

Ruscica

Moriham

and

Tsuzuki

(1969)

have

also

a-chymotrypsin,

was

hydrolyzed

role

1968).

some

a specific

been

for

the essential

are

only

For

of a

They

exhibit

L-phenylconflicts

between

inhibitor

of a-

(1968) found

proteolytic

subtilis).

os L-tyrosine,

there

to TPCK,

the

of Bat.

Ruscica,

such

However,

insensitive

between

to be inhibited

that

Bz-Tyr-NH2,

a small

amount

by sub-

using

various

synthetic

BPN’. To clarify

the

peptides

such

(A or

B = various

trypsin

BPN’

similarities

has recently

ZPBK.

substrate

mechanistic

residue

aromatic and

are

subtilisin

in both

Subtilisin

chymotrypsin,

there

serine

against

etc.(Morihora

them

split

and

specificity

alanine,

that

at the and

conflict,

a systematic

as Z-A-Tyr-Nh,

peptide

amino

* The obbreviations tosylamido-2-phenylethyl chloromethyl ketone; Abbreviated designations IUPAC-IUB Commission

was

Z-A-Gly-Tyr-NH*, acid

bond

subtilisin

study

BPN’.

residues)

containing Therefore,

undertaken Z-Tyr-B-NH,, These

as substrates. the

corboxyl we

should

group

synthetic

of L-tyrosine

be able

to examine

and

Z-Tyr-Gly-B

peptides

should

by both

a-chymo-

the

effects

be

of the

used ore: DFP, diisopropyl fluorophosphate; TPCK, L-lZPCK, benzyloxycarbonyl-phenylalanine chloromethyl ketone; ZPBK, benzyloxycarbonyl-phenylalanine bromomethyl ketone. of peptides or the derivatives obeyed the tentative rules of the on Biochemical Nomenclature.

210

Vol.

35, No.

neighboring

residues

varying

the

sizes

BIOCHEMICAL

2, 1969

and

kinds

the

previously

specifici

and

end

amino

occupying

subsites Our covers

S2’,

results

amino

properties to the

This

in both

lined

and

occupies

occupy and

the

adjacent those

are

in such

same

location

subsites,

those

towards

the

by on the

if the assumptions

(1967)

enzyme

the

substrate

an information

enzymes,

Berger

COMMUNICATIONS

peptide

provide

up on the

always

S,, S2, etc.,

of the

would

Schechter

are

residues

indicated

that

18 A and

acid

can

residue

of the tyrosine

L-tyrosine

sites

by

RESEARCH

true.

These

a way

that

the

(the

ca)alytic

towards

the

COOH-end

NH,-

occupying

etc.

at least

one

acid

sensitive

active

substrates

subsites

St’,

of the

to be hydrolyzed

2) the

BIOPHYSICAL

peptides.

papain

1) the

linkage

site)

ties

concerning

are;

CO-NH

the

of A or B in the

made

assumptions

surrounding

AND

both

be divided

of peptide

large

residue

enzymes

have

into

at least

substrate.

active

sites

of the

peptide

a considembly 5 “subsites’,

Further,

of the

enzymes

active

each

are

subsite

that

S,, which

considerably

site

which

accomodating

it was ascertained except

substrate,

large

the

lies

different

adjacent

from

one

another. MATERIALS Some

peptides

laboratory, was

the

checked

specified,

by elementary the

constituent

a crystalline Co.,

mixture

containing

volume tilisin

(for

Osaka.

law

of 1 ml BPN’).

The

solubility (or 5 ml), enzyme

and

be reported

analysis

and

by thin

acids

from

incubated

concentration

others

in an another

Their

paper.

purity

of the

L-configuration.

A crystalline

Corporation,

(subtilisin

determined

a suitable

amount

(for

a-&ymobypsi

adjusted

Jersey,

was obtained

as follows:

substrate,

when

New

BPN’)

3 mM

was suitably

211

in our

Except

was

at 30”

prepared

chromatography.

subtilis

and

were

layer

of pH 8.5,

peptides),

the

Biochemical

activity

M Tris-buffer

was

all

of Bat. --

peptidase

of mast

were

Worthingtdn

proteinase

0.05

The

commercially, will

amino

alkaline

METHODS

of which

was obtained

Nagase

amide

obtained

procedures

a-chymotrypsin and

were

AND

15%

A reaction dimethylform-

of enzyme, n)

or

from

40”

to determine

in a final (for

the

sub-

initial

Vol.

35, No.

2, 1969

rate

of proteolysis.

was

withdrawn

BIOCHEMICAL

At every and

to stop

further

method

of Yemm

compounds

put

into

The

DNP-method.

tube

sites

isomeric were

I shows

Ammonia

released

peptides,

acid

they

as had residues

PZ, etc.,

to as PI’,

Pz’,

specificity which

the

catalytic

The

latter

case

chymotrypsin

in

Table

indicate

II.

amidase bond,

subtilisin subsite,

as will

BPN’. while

be seen

The

reactivity

the

reverse

are

were

compounds,

using

Conway’s

enzymes

against

carboxyl

various

group The

numbered

at all The

table. the

in the enzyme

case

according

subsite

both

to act

as a

of subtilisin is seen

the

of the

as the

same

residue

rather

BPN’

is greatly

that

of a-

substrate. as the

a-chymotrypsin

sensitivity

of

as a depressor

for

by L-alanine

at

promoted A more

BPN’.

for

to the acts

distance

between

of hydrolysis

be ascribed

ST’-S,’

determined

is observed

can

212

and

than

was

referred

a stereo-

peptide

theenzymes

referred

are

53 of subtilisin

difference

in a-chymotrypsin.

etc. that

site

subsites

are

as S,-S,

active

promoter

of the

decreases

of subsite

diastereo-

to the

S2’,

such

however,

N-terminus in

St’,

subsi tes,

has a larger

to the

but

by

of L-tyrosine

positions

It indicates

specificity,

Z-Tyr-Tyr-NH1

later),

deduced

apparatus.

in the

seems

authentic

or by the

as shown

S,, a remarkable

against

substrates

to subsites

the

N HCI

by using

adjacent

of each

activity

upon

made

those

corresponding

residue

of 0.1

to subsites St, S,, etc.

are

except

As to subsite

were

specificity.

substrate

St.

that

region

An aromatic

poor

Tyr-Tyr

increases

side-chain-specificity

enzymes: (the

may in the

The shown

site

1 ml)

mixture

by the ninhydrin

measured

the

their

enzymes

subsite

(or

adjacent

and

central

of both

from

which

the

ml

reaction

DISCUSSION

containing

peptide

residues

in both

the

AND

COMMUNICATIONS

of the

authentic

was measured

expected

respectively,

is present

from

been

was

comparing

linkages

respectively,

etc.,

surround

the

0.1

of enzymes

activities

(P) in the

i .e *, the

occupy,

to as Pt,

the

in which

hydrolyzed

amino

proteolytic

1 ml) each

calculations

of action

hydrolysates

RESEARCH

contained

in which

of the

the

(or

of hydrolysis

RESULTS Table

ml

which

(19.54, The

BlOPHYSlCAL

0.1

extent

Cocking

chromatogram

usual

a test

as a standard.

a paper

3 minutes,

hydrolysis. and

AND

striking

effect

is

Vol. 35, No. 2, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

I.

Stereo-specificity

The arrow shows the bond split. Peptides (a) were obtained Corporation, and those (b) were prepared in this laboratory. Peptide P,---Ps

-

P,‘-

Table Peptides

(a)

Center”

and

(b) were

at Osaka Pepti

p4-

Z ZzZ Z-Gly-Gly-Tyr-NH, Z -Ala -

observed seen not

observed The

Side-chain-specificity

described

in Table

Gly zZ Z Z Z Z Z Z z-

-

correlation

Peptides

BPN’ )

0.920 0.008 15.200 0.067 0.005 0.000 0.010 0.004

(c) were

obtained

from

University.

de

a-Chymotrypsi n ( pM/min*mg

-P,’

Tyr -NH, Tyr -Gly Tyr -Ala Tyr -Ser Tyr -Leu Tyr -Tyr Tyr -Phe Tyr -Gly Tyr -Gly Tyr-Gly-

of the

peptide

of subsite

NH2 (a) NH2 (a) NH, (a) NH, (c) NH, (a) NH2 (a) Gly (a) Ala (b) Phe (6)

chain

to the

Z-Gly-Tyr-NH2

Subti lisi n BPN’ enzyme )

0.011 0.186 0.067 0.031 0.020 0.000 0.048 0.177 0.051 0.358 0.297 0.488 1.092 0.208 0.007 0.014 0.015

(c) (c) (c) (a) (a) (a) (a) (b) -

between

i n a-chymotrypsi

specificity

I.

Chemical

Subtilisin enzyme

0.031 0.005 0.177 0.034 0.358 0.010 0.014 0.004

NH, (a) NH:, (b) L-Ala (b) D-Ala (b)

AC -Tyr -NH2 Bz -Tyr -NH, Gly Tyr -NH, Ala -Tyr-NH, His Tyr -NH, Tyr Tyr NH,

by elongation

in the

(a) (b) (b) (b)

II.

--pl+h

p3 -p2

sin (I-’ AJ minsmg

P2’

L-Ala -Tyr NH, zD-Ala-Tyr NH, L-Ala -GlyTyr NH, D-Ala-GlyTyr NH, Z -Tyr -L-AlaZ -Tyr -D-AlaZ -Tyr -GlyZ -Tyr -Gly-

“Peptide

Cycle

a-C hymotry

p2 -p,+

z-

Z Z -

from

N-terminus and

0.011 0.003 0.098 0.920 0.017 0.033 5.000 15.200 0.004 0.005 0.000 0.016 0.000 0.000 0.010 0.010 0.050

in subtilisin

Z-Gly-Gly-Tyr-NH2,

BPN’

as

but

this

n. 5,’

is much

more

213

strict

in a-chymotrypsin

than

in sub-

is

Vol.

35,

No.

tilisin

2, 1969

BPN’;

does

L-tyrosine

glycine

amino

BIOCHEMICAL

residue

Z-Tyr-Tyr-NH, bond

ascribed

In this

a considerably

such

as S,-S,

amino

acid

residue

taking

3.5

ft p er one

subsite

St in both

various

L-amino

n.

The

enzymes

The

degree

of the

of subtilisin

BPN’

site

at both

sides

from

enzymes

were

may them

difference

of a-chymotrypsi

n.

that

bond.

thus

are

there

of the

large

than

that

5 ‘subsites” one

as about

using

and

of the enzymatic

(X = , 1969).

than

remarkable

of subsite

of

Tsuzuki

specificity

site

18 8,

Ac-X-OEt

hara

active

be

BPN’

The specificity

(Mori

that

might

accomodating

calculated

determined

that

of a-

differences except St.

The

properties

between

subsite

S,.

X-ray

analysis

of the

large

REFERENCES Moriham, Shaw, Schechter, Yemm,

K. E.,and

and

Tsuzuki,

Ruscica, I.

E. W.,

and and

H., J.,

Berger, Cocking,

Arch.

J. A.,

Biol. Biochem.

E. C.,

Biochem. Chem.,

Biophys., -243,6312

Biophys. Analyst,

so,

214

(1969)

.

(1968).

Res.

Commun.,

209

(1955).

in

the

subtilisin

into

each

substrate.

paper

split

enzymes.

and

site,

are

specific

are

This

the

at least

catalytic

than

hydrolyzes

in both

broader

be greater

the

St’

degree

by any

bonds

Tyr-NH;!

a somewhat

indicated

may

peptide

be divided

comparatively

properties

clarify

can the

higher

a-chymotrypsin

of peptide

showed

study

difference

both

in an another

BPN’

enzymatic

the

The sizes

residue

as reported

present

in their

from

substrate. acid

subtilisin

the

active

which

amino

acids),

that

site

times

a-chymotrypsin

splits

that

COMMUNICATIONS

of hydrolysis

The

of subsite

active

of peptide

the

BPN’

we reported

St ‘-S2’

promotion

the enzymes;

subtilisin

RESEARCH

twenty

BPN’.

of the specificity

large

and

It indicated chymotrypsi

and

communication,

have

no such

by both

difference

by about

in subtilisin

different

of Tyr-Tyr

to the

while

is observed are

BIOPHYSICAL

hydrolysis

in a-chymotrypsin,

acid

peptide

promotes

AND

1_7, 157

(1967).