Separation of polar, steric and specific effects in the α-chymotrypsin-catalyzed hydrolysis of acyl-substituted p-nitrophenyl esters

Vol. 4 1, No. 2, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SEPARATION OF POLAR, STERIC AND SPECIFIC

EFFECTS IN THE cr-CHYMOTRYPSIN-CATALYZED

HYDROLYSIS OF ACYL-SUBSTITUTED Alain

Oupaix,

Laboratoire

Jean-Jacques

d'Enzymologie

Faculte

p-NITROPHENYL

BQchet

and Colette

Physico-chimique

des Sciences

d'0rsay

ESTERS. ROUCOUS.

et Moleculaire

- 91 - Orsay,

France.

Received September 15, 1970 SUMMARY. The a-chymotrypsin-catalyzed hydrolysis of various acyl-substituted p-nitrophenyl esters has been studied at 25'C, between pH 6 and 8. The substituent effects on the deacylation rates of corresponding acyl-chymotrypsins have been analyzed using the Taft-Ingold relationship and contributions of polar, steric A specificity constant S has been defined and and specific effects, separated. its value discussed in relation to the structure of the substrate.

Use of linear complicated series

by the

substituted

nature

values

alkyl

substrate

to the

polar,

steric

R on the rates

5Further

work

of deacylation’

on the

acylation

relationship. p are

with

variable,

chain (61,

of different

in the

is

order

and

now in progress

of Nreactions,

depending

on the

reaction.

The aesters

with

of reactivity

(5,61

(5,6,71. we have tried non-conjugated

a-chymotrypsin-catalyzed

464

of m&z-

p-nitrophenyl steric

relationship

effects

step

a long

to some

For these

of the enzymatic

shows a normal

of derivatives

limited

is

('21 and anilides

of acyl-substituted

on the a-C,

and specific

step

catalysis

hydrolysis

benzoylimidazoles Hammett

and the studied

By use of the Taft-Ingold the

enzymatic

and has been often

constant

hydrolysis

groups

the exception

obeys

(1 I,

of the reaction

chymotrypsin-catalyzed various

in

So, the a-chymotrypsin-catalyzed

c3.4)

of the

relationships

of enzymes

phenylacetates

acetyl-L-tyrosine experimental

energy

specificity

of substrates.

para-

with

free

to separate substituents

hydrolysis

in our

laboratory.

of

Vol. 41, No. 2, 1970

BIOCHEMICAL

acyl-substituted

p-nitrophenyl

specific

deviations

in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

esters, the

R-COOC6H4pNO

free

linear

energy

TABLE

a-chymotrypsin-catalyzed

hydrolysis

2

,

and

to

analyze

particularly

relationship.

I

of

paranitrophenylesters

acyl-substituted

R-COOC6H4pN02

:: R I ( [ --------------i

: :

PK;' -------------;

(al:

k3,x

kg

I

x[bl:

-1 ; ,02sec-' IO'sec expl. calcul.: -------------i----------:----------i----------j___________l

;

E

u

S

x

I 1 1 1

S

; Cl-CH2

;

6.90

f

0.04

;

262

*

12

;

230

;

-0.24

;

1.05

j

0

(H

:

7.60

f: 0.09

:

150

f

25

:

140

:

+I.24

:

0.49

:

0

;I-CH2 (CH3-0-CH2

i :

6.95 7.16

* 0.05 * 0.02

i :

;Cl-(CH212

i

7.13

* 0.01

C C6H4-CH2

:

7.41

ICH3

;

7.40

t (~ii~l~cti

:

; [CH313C Cl-(CH213

64

:

-0.37

;

0.85

1

0

28

:

-0.19

:

0.64

:

0

2.14

;

-0.9

i

0.385

;

0

2.05

:

-0.38

:

0.215

:

1.21

i

0

:

0

0.196

:

-0.47

:

-0.19

0.018

i

-1.54

;

-0.3

:

-o.35c

:

;

: .

-o.35c

i

* 0.5

:

:

-0.38

:

0.08

* 0.6

i

:

-0.45

i

0.02

;

1.275

*

:

:

-o.45C

:

* 3 * 9

:

i2.09

* 0.03

j

* 0.02

:I.08

* 0.05

:

f

0.01

io.99

* 0.015

;

7.34

f

0.02

:0.34

i

:

;

7.04

f

0.02

b.018

* 0.002

i

:

7.33

* 0.02

:5.18

* 0.15

:

Cl-(CH214

;

7.41

* 0.06

j4.45

* 0.37

~c~H~-[cH~I~

:

7.26

* 0.02

:17.8

iC6~4-(~H2)3

;

7.33

*

0.05

jl2.0

(C6H4-[CH214

:

7.73

a+ 0.05

:I.85

: Z-Gly

;

7.01

* 0.03

:

[Z-L-Ala

:

7;21

f

0.02

iI89

'4

[ I Z-L-Leu

;

7.59

*

0.04

j670

*

40

i

i-2.3

*0.3c;

:

7.53

* 0.06

:3700

*

300

:

:-2.3

'0.3':

(Z-L-Phe ( Conditions

57 34

35

0.08

0.14

+ 1 :

0 0

:

0

:

0.555

1 1 1 1 1

:

0.69

I'

:

1.26

1 I )

0

0.14d 0.051

I 1 1 I 1 I 1 1 I I

d:

O.Ozd

:

0.465

; -o.45*o.lCZ

0.57d

0.46

:-1.25'0.Zci

0.46d

: : :

0.4Zd

;

3.44

0.51sd

:

3.965

1 I I I I ]

2.02

I I

: :

T

[a) [bl

[cl cdl

= 25'

C, I = 0.5

An iterative constants The

(NaC11,

fitting k

theoretical

from the values The E values compo?nds with The ux values and corresponding

% (v/v1

of the data

at

3.x'

4.5

optimum values

on

px= 2.35 estimated

and

k3,x of

were similar were ui

pH.

size

calculated (151.

to and

equation KY.

the

correlation

6 =

after (81. from

465

acetonitrile-water. 1 gave

line

the

were

deacylation

recalculated

0.73.

comparison

with

known

E

values

of

S

pK

values

of

the'conjugated

acids

Vol. 4 1, No. 2, 1970

MATERIAL

BIOCHEMICAL

AND METHODS

The esters

of p-nitrophenol,

the dicyclohexylcarbodiimide

spectrum

of their

previously

hydrolysis

acids

a Worthington,

concentration a molar

are products

at 25°C.

was found

in buffered

water.

from

Employed

equal

of 0.025

trophotometrically with

a Cary

The molar 16,300

analysis

product

no CD1 613761.

weight

(121

measurements

The hydrolysis

the

% (v/v)

16 spectrophotometer

coupled

ion

was determined

equal

to 7.03

of out

pH 6

pH 7 and 6, at

studied ion

Graphispot

was measured

percent

acetonitrile-

between were

of the

between

of p-nitrophenolate

to a Sefram

of p-nitrophenolate

; pK of‘p-nitrophenol

acid

IO4 Ill).

carried

acid

reactions

appearance

: the

were

4.5

'-Z-ethane-sulfonic N (131.

of 2.5

by measurements

[NaCl),

Its

at 260 nm, using

and molecular

I = 0.5

case of

Company.Chymotrypsin

(batch

was titrated

the U.V.

of N-a-C-carbo-

spectrophotometrically (IO)

They

in the

esters

by

use.

spectrum,

Z-(N-morpholinolethane-sulfonic

by following

absorptivity

U.V.

Chemical

%. Kinetic

solutions,

were

before

p-nitrophenylacetate

to 92-94

and 7, N-Z-hydroxyethylpiperazine-N the concentration

free

sites

using

aqueous

buffers

the Pierce

salt

of active ion,

their

The p-nitrophenyl

of 50,000

concentration

site

point,

was determined

coefficient

1, have been prepared

and elementary

compounds.

of p-nitrophenolate

active

melting

3 x crystallyzed,

extinction

in Table

(91 and recrystallyzed

products,

in solution

The absolute burst

by their

non described

benzoxy-amino

given

method

have been characterized

is

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

specat 400 nm,

enregistror.

and found under

our

equal

to

experimental

conditions. RESULTS. Between site

ionizes,

phenyl

the mechanism

esters

E 5

pH 6 and 8, in the pH range

11 EH

+

by the

-> k2

S;'ESKS K; '11 S -

the

histidine

of the a-chymotrypsin-catalyzed

may be represented

+

where

EHS

hydrolysis

scheme I (141

ES' K; 11 EHS'

k3 .-)E + '1

57 of the

active

of p-nitro-

:

+

p2 Scheme I

BIOCHEMICAL

Vol. 41, No. 2, 1970

where

S is

the

substrate,

and

EHS

ES

enzyme, ES'

and

EHS'

are

the

hydrolysis In

the

E and

the

EH

are

corresponding

different

steady

the

active

ones

forms

products,

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

of

the

the

inactive

conditions

S,>>

the

P,,

;

and

carboxylic

and

where

of complex

intermediate.

p-nitrophenol

under

forms

enzyme-substrate

acyl-enzyme

respectively

state,

and

E,

and

S,

P2

acid. 5

> K m aiw

the

appearance

rate

of

P,

given

kg

and

by

the

equation

(11

:

(11

dt

1

+ H/K;

The

experimental

values

The

Taft-Ingold

relationship

of

substituents

R on

effects

is

k+

-= dP1

of

product

p-nitrophenyl

esters

Ak3 x

log

of

has

rate

the

px 0x

+

are

been of

R-COOC6H4pN02

=

K;'

given

used

in

to

Table

1.

evaluate

the

different

a-chymotrypsin-catalyzed

hydrolysis

:

6E

(2)

S

k3.0

In

for

any

and

Es

equation

acyl-enzyme

are

:

polar

steric

reaction

Taft's

tables

5

The

kg

kinetic

o

I

is

(81,

best 373

apparent

for

but by

the

The

standard

some

correlation

has

'In

substrates,

order to obtain water and in 70 our laboratory. in

and

precise % [by

S,

p*

ones.

and

K was

varied

constants, volume1

467

for

the form

was

determined

the

aqueous

alkaline acetone

6, and

;

polar

the

ox and

are given

Es

were

constants

has

between

R = CH3

in

estimated

A

calculated

been

rate

deacylation

and o*

these

similar

the

for

constants

m aw studied

is

constants,

calculator

constant

x

9

comparison

substituents, with

Michaelis

of

substituent

comparison

electronic

k3

constant

substituent

constants.

The model

the

steric

and

approximatively

Wang

(21

nine

compounds

M and

is

a

:

<

'IO -4

with

hydrolysis under

5.10

-6

M for

all

10m5M.

of investigation

these

esters in

Vol. 41, No. 2, 1970

log

k

log

k3

3 I x’k3

I0

, x’k3

,0

substituent on

[or pi1

0.051E

-

CO.76

t

0.041E

and

the

s

8

defined

by

=

c2.35

=

(5.5

vary

line

comparable

of

acyl-substituted of

f 0.0610*, f

0.2la

(151.

over

In

a wide

to values

form

in

figure

range

of

1.5

in

or phenyl base

equivalent

one,

Us is 1,

polar

104.

obtained

of

the

which

the

of

ethyl

some

or

i,

of reactivity

6 are

involvement

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Charton

to a spread

hydrolysis

suggest

mechanism

f

leading

catalyzed and

(0.73

correlation

the

requirements p*

-

constant

tuents

of

BIOCHEMICAL

polar

the

the

substi-

and

steric

values

Experimental the

hydroxide

esters

catalysis

in

ion-

(8, the

16.

171.

deacylation

[21.

Z-L-Phe

Z- L-Ala f

/.

kH,

Figure

1 : Representation

.

.

.2

.4

.6

Taft-Ingold

relationship of. ac.yl-substituted

of

the

.

trypsin-catalyzed hydrolysis esters, R-COOC5H4pN02.

x are

.

.6

.

1.

I+

for

the

a-chymo-

paranltrophenyl-

= 25OC, I= 0.5 (NaCl), 4-5 % [v/v) acetonitrilewater. rate constants for deacylation at optimum pH. The

the

correlation

line

to

2.

equation

.

: T

Conditions k3

.

was obtained

468

by an iterative

fitting

of

the

data

Vol. 41, No. 2, 1970

BIOCHEMICAL

A number and the polar

reactivity

effects

to include

k3 x/k3 o , .

chain

A similar

value

phenomenon

by different

the

for

(18,

alkyl

greater

than

a chain

length

5-7,

191.

may explain

the

effect

of the

chymotrypsin-catalyzed

hydrolysis

apparent

for

: the

S value

R = CSHS(CH214, However,

which

these : so,

in the

(31

:

line

the spelength

i,

then

with

decreases

analogous

of S for

with

aliphatic

beyond.

compounds

the more hydrophobic value

of

model1 , increa-

extended

character

compounds

with

compounds

where

requirements atom,

for

work

and asymmetric

group

-O-CONH-,

R

has a comparable carbon

chain

similar

to that

is observed

length.

atom becomes important

asymmetric

deviations

than

in the c-

of p-nitrophenol

R = CSH5CH20CONHCH2 is

are much larger

(20-241

have previously

a-chymotrypsin

acylaminogroup

is

acyl-amino

those

for

as in N-a-C-

occur. linear

The values long

case of N-a-C-carbobenzoxy-L-phenylalanine,

authors

In order further

of IO-II

larger

derivatives,

derivatives

to 4. Numerous

carbon

equation

derivatives,

of N-acyl-glycinate

when the alpha

carbobenzoxy-L-aminoacids

ture

has to be

or Cl(CH214.

No specific

corrpounds

than

and the correlation

a fully

Moreover,

or CSHS(CH213 in comparison

of S for

other

some particular

observed

R = CSHSICH212

for

factors

11.

0 beyond

assuming

ring

Cl(CH213

line,

equation

or phenylalkyl

of the benzene

is

points

S [see table

has been previously

authors

that

S, following

experimental

(8 i approximatively

ses to an optimal

implies

correlation

p* c* (31.

case of linear

S becomes

the

The Taft-Ingold

constant

of the constant

constant

alkyl

=

from

compounds

important.

between

So, in the

the

are

- 6E S + S

a measurement

cificity

significantly

a specificity

The distance gives

deviate

of corresponding

and steric

modified log

of points

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

specific

and alkyl

compounds

which

of the

in our don't

substrates

or phenylalkyl

to make the meaning now in progress

emphasized

specificity

laboratory

carry 469

S is the

following

: asymmetric chain

branched constant

upon D-aminoacids

an acylaminogroup.

chain near strucalpha on it.

S precise derivatives

Vol. 41, No. 2, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS.

grateful

We

would

like

to

Prof.

J.

to Yon

thank

Or

for

her

8.

Capon

for

interest

in

helpful

this

discussions.

We

are

work.

REFERENCES.

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