On the size of the active site in proteases: Pronase

BIOCHEMICAL

Vol. 46, No. 5, 1972

ON THE

SIZE

OF THE

ACTIVE

Elvin From

the Department

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SITE

Harper*+

IN PROTEASES:

and Arieh

Berger

Weizmann Israel

of Biophysics, Rehovoth,

PRONASE

Institute

of Science,

Received February 11, 1972 Summary The size of the active site of the enzyme Pronase has been determined by the method of diastereoisomeric replacement and shown to be five amino acid residues in length. The application of this method for measuring the size of the active site to broad spectrum enzymes is demonstrated. In continuation active

site

a broad

of Pronase

substrate

proteins.

The

method

which

modifications of their

further

alteration

’ Supported

acid

if this

enzyme

would

the size

of the

is an enzyme

enzymic

with

hydrolysis

adhere

examined

compares

of

to the rules

(3) then one could to investigate

the activity

of

utilize

the active

of the enzyme

and diastereoisomeric

residue

has alter

from

no longer

by N.I.H.

total

replacement

length

distance

Address: Hospital,

Pronase for

previously

employed

in structure

function

* Present General

that

(i),

in general.

of increasing an amino

series

investigated.

of diastereoisomeric of proteases

in this

employed

enzymes

size

peptides

(2) was

thought

specific

the method

studies

specificity

It was

the more

site

of previous

been

replaced

of reaction

the point

of cleavage

the rate

Developmental Biology Boston, Massachusetts Fellowship

1956 Copyright 0 1972, by Academic Press, Inc.

by its

the rate

affects

Postdoctoral

pairs

antipode.

in

These

enzyme

as a

by the enzyme.

When

of reaction,

fF

of peptides

of the

Laboratory, 02114.

towards

the length

Massachusetts

2AM-34-222-01

of the

Vol. 46, No. 5, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

This

active

site

has

been

measured.

point

to be

the

same

at

of

this

point

all

of cleavage

times

to affect

peptides

Berger

(4).

purified

by

were

Pronase

the

the

method

of

examinedbyquantitative

as

described

O.O3M,

Pronase

and

0.05M

A

k value

are

given

in

The

k values

assuming site.

acid ing

order

to the

distance

was

only

doubled

rate

from

the

an

increase diminishing

reaction

contained pH

substrate

7.8

in

one

ml.

of bonds vary

substrate cleaved

enzyme

in

the

catalytic

in

subsites

and

the

catalytic order

were

and

can

the

a D

be

at

substrate

by

the

enzyme

is always

The their

residue.

interpreted are

site. exert

of alanine

with

aminoacids

is

first

peptides

remaining

at amino

influence

accord-

site.

with

regard

obtained

when

to the

enzyme

and

substrate

sub-

concen-

tripled. increases

200

dipeptide

demonstrating is

cleavage

that

constants or

The

electrophoresis

CaCl2

bond

was

constant

L residues

tripeptide

rate

no

from

further

Discussion

of the

are

studied

(5).

reaction

constants)

adjacent they

reaction

The

the

occupy

identical

tration

array

peptide of

rate

and

and

on 20 diastereoisomeric is

side

of Schechter

BioChem

Narahashi

0.005M

and

There

a linear

residues

strate,

trating

(first

region

The

There

1.

The

same

Pronase

Table

that

active the

for

method

Gal

typical

Tris

Results The

the

highvoltagepaper

(3).

2Oy,

oneither

Methods

from

Nomato

hydrolysis

rates.

by

obtained

productswere previously

and

the

changes

reaction

synthesized

was

assumes

andintroduces

Materials Alanine

method

the of

to

effect

of

in

the

tripeptide

effect

2. 5 fold

fold

and

of increasing from

tripeptide

additional

1957

subsites

case

of peptides

500

fold

from

the

peptide

to tetrapeptide on

enzyme

containing dipeptide length. illuscatalytic

to

Vol. 46, No. 5, 1972

BIOCHEMICAL

AND BIOPHYSICAL

Table Pronase

Cleavage

Peptide

RESEARCH COMMUNICATIONS

1

of

Relative

Alanine

Peptides

Rate

k t min’

L-

LLL

100

0.36

80

0.30

L-LLLD

70

0.25

L-

LLD

60

0.20

L-

LLDL

50

0.20

L-LL

40

0.15

DL-LL

20

0.08

15

0.05

DLLL-L

5

0.02

L-LDLL

5

0.02

L-

5

0.02

L-LD

5

0.02

LDLL-L

2

0.008

DLL-L

2

0.007

LDL-L

0. 7

0.003

DL-LLD

0.5

0.002

DL-L

0.3

0.001

L-L

0.2

0.0007

DLLD

0

LDL

0

DL-

LLL

DL-

LLLD

LDL

rate zyme. equals

as

the This bond

substrate effect cleaved)

approaches of increased

the

size

peptide

:

1958

of length

the

active is

as

site follows

of

the

(dash

9

en-

Vol. 46, No. 5, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

k 100 40 0. 2

L-LLL L-LL L-L Maintaining

the

peptide

D demonstrates the

the

D residue

length role

This

to the

is

and

changing

of diastereoisomeric

adjacent

breakage.

constant

seen

in

L residue

replacement.

susceptible the

the

bond

tripeptide

lowers

series

to

Placing the

rate

of

bond

:

k L-LL L-LD DL-L LDL the

tetrapeptide

series

40 5 0. 3 0

: k 100 60 20 5 2 0.7 0

L-LLL L-LLD DL-LL L-LDL DLL-L LDL-L DLLD the

pentapeptide

series: k 80 70 50 5 2 0.5

DL-LLL L-LLLD L-LLDL L-LDLL LDLL-L DL-LLD The

enzyme

assumed

size

an L residue binding

of the

the

on

of

its

usually

Inspection sites

favors

the

substrate.

substrates

that

active

site.

causes

a dimunitionin

contain

Introduction

all

L peptides

within

of a D residue rate

probably

there

are

in

due

to

the

place

of

decreased

substrate. of these enzyme, Assuming

data each

suggests

that

of whichwill the

size

combine of each

1959

residue

with

five

stereospecific

one

to be

3.5

amino A,.

acid This

of is

Vol. 46, No:~,

in

the

BIOCHEMICAL

1972

range

of

the

size

carboxypeptidase

A

AND BIOPHYSICAL

determined

by

this

RESEARCH COMMUNICATIONS

method

for

papain

(1)

multiple

and

(6). Conclusion

Pronase, site

a broad

attachment.

divided

The

into

These

subsites

data

grooves

spectrum

are

in

in

which

length with

of

the

with

substrate

binds

to

peptides

by

site

is

5 amino

acid

active

differences

keeping the

enzyme

in

that

their

of work

sets

binding on

residues

characteristics.

enzymes

possessing

(7).

ACKNOWLEDGEMENT

The peptides

authors

thank

employed

Dr.

in

Israel

these

Schechter

for

the

gift

of

the

alanine

studies.

REFERENCES

i.

Schechter, -27,

157

I.,

and

Berger,

A.,

Biochem.

Biophys.

and

Y.,

J.

Biochem.

Nomato,

3.

Schechter,

I.,

and

Berger,

A.,

Biochemistry?,

4.

Schechter,

I.,

and

Berger,

A.,

Biochemistry

5.

Nomato,

6.

Abramowitz,

7.

Blake, Sarma,

M.,

Narahashi,

2.

Res.

Res.

GommUn.

(1967).

and

M.,

N..

Commun. G. G. F., V. R.,

Narahashi, Schechter, -29,

Mair, Proc.

862

Y., I.,

J.

andBerger,

-46,

5,

Biochem. A.,

653

(1959).

3371

(1966).

3362

(1966).

-46, Biochem.

148 i

(1959).

Biophys.

(1967).

G.A., North A. C. J., Phillips, Roy. Sot. (London) 13167, 365

1960

D. G. (1967).

and