The hydrolysis of O -hippurylglycolate catalyzed by carboxypeptidase A. Evidence for possible allosteric effects

BIOCHEMICAL

Vol. 21, No. 5, 1965

THE HYDROLYSIS PEPTIDASE

OF -0-HIPPURYLGLYCOLATE EVIDENCE

A.

Department

October

In the

the

only

of the

studies

we have

fits

complicating products, in

substrate

inhibition

two kinds

terms

factor

competitive

substrate

analoqous

to that 2

hippuryl-L-mandelic

plot

of

acid.

for

this

for

CarboxypeDtidase

compound

represented v,/(E)~

The empirical

of the

vs.

these

in Fig. (S),

equation

x 107 1.2/moles2/min.

is v,/(E),

inhibition.

(uncorr.), the data

is the

A lacking

1.

2s3 To we now

observed

in tne

was synthesized

preparation

indicative = a(S)2 and b = 7.8

of -O-

determined first

at pH 7.5

reported

ester

an asymmetric

As can be readily

444

can be

noncompetitive

we have

used for

which

by one

of g-hippurylglycolate.

165-66"

The rate

latter

substrates,

which

hydrolysis m.p.

of scheme,

inhibition

product

for

in

behavior

Michaelis-Menten

of both

activation,

acid,

are

and that

and competitive

&Hippurylglycolic

atom

being

of a combination

A-catalyzed

substrate

The hydrolytic simple

found

observed

of g-acetyl-L-mande-

a relatively

carboxypeptidase

1.39

192

of behavior

add a third,

the kinetics

hydrolysis

L-mandelate,

interpreted

25.0"

of Chicago

analyzed

A-catalyzed

ester

in a manner

EFFECTS

F. W. Carson

and

University

and g-hippuryl-L-mandelate. former

and

Awazu

ALLOSTERIC

27, 1965

recent

the

the

S.

of Chemistry,

carboxypeptidase

late

CATALYZED BY CARBOXY-

FOR POSSIBLE

E. T. Kaiser,

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

substrate

carbon

seen,

the

activation.

+ b(S),

where

x lo4

l./mole/min.,

a =

Vol. 21, No. 5, 1965

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

7. .c E -0 iii ;: >O B-i ‘0

Fig.

1.

v,/(E),

~0

glycolate

WC

f or the

Hydrolysis

of +Hippuryl-

Catalyzed

by Carboxypeptidase

5

IO I03qp)-+

A at

pH 7.5,

25O.

Fig.

2.

v,/(E),

vs.

glycolate 25'

in the

(S), Catalyzed Presence

for

the

15

Hydrolysis

by Carboxypeptldase of 0.05

445

20

of c-HippurylA at pH 7.5,

--M N-Carbobenzyloxyglycine.

BIOCHEMICAL

Vol. 21, No, 5, 1965

holds

over

to more

the

than

It

Is

seen In

substrate 1 x lo-'

to suggest

hydrolysis

carboxypeptldase of this

A may have

Fig.

activating

OxypIyclne

0.05

Fig.

the

rolysis

found

1 Is no longer

in

2.

rate

In

the

of the

7 x 10m4 to 2 x 10-Z E.

strates sterlc. involved strate

in

found

the

formation

complexes

is not

A-catalyzed

of

If

sterlsm

of

associated

have been moderate acter?

Physical

further

information

In

of 0.05

activation

addition

of an as

--M N-carbobenzylA-catalyzed

the

equation

similar

enzymes

hyd-

v,/(E)~

recognized

of

sub-

to be allo-

between

the

sites

or enzyme-subit'ls

not

possible

activation

phenomena

hydrolysis

of &hlpourylglycolate

changes

=

concentrations

to effects

relationship

A 1s indeed questions

with forms?

reported5 molecular

the

support

in the

found

to

in

enzyme

the

or

factors.

intriguing

polymeric

the

the

carboxypeptldase

a number

are

of conformational

other

after

known at present

carboxypeptidase

because

substrate

of enzyme-activator

whether

because

that In

at substrate

with

since

say conclusively

result

the

presence

observations

Of course,

means

that

x 105 l./mole/min.

and modifiers 4

activation

character.

obeys

from

kinetic

substrate

carboxypeptldase

where

These

3 x 10 -4

from

M N-carbobenzgloxyglyclne, --

of &hippurylglycolate c = 2.7

the

apparent

c(S)

with

that

allosteric

we have

modifier,

indicated

range

of &hlppurylglycolate

possibility

shown in

concentration -M.

tempting

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the Also,

for weight

monomeric are

state

the

Indicative

concerning

enzyme,

can be raised.

some other

and chemical

an allosterlc

our laboratory. 446

of the

cooperative

proteolytlc

the

effects

allosterlc

designed

questions

allo-

enzyme

enzymes

of their

experiments these

Is

are

then

or which

of char-

to provide in

progress

Vol. 21, No. 5, 1965

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Acknowledgement This Institute Health

work was supported of Arthritis

by a grant

and Metabolic

from

Diseases,

the

National U. S. Public

Service.

References (1)

E. T. Kaiser

2922 (1964)

and F. W. Carson,

J. Am. Chem.

Sot.,

86,

l

(2)

E. T. Kaiser and F. W. Carson, Comm., 18, 457 (1965).

(3)

E. T. Kaiser, results.

(4)

A leading

(5)

C. G. Trowbridge, Biochemistry, 2,

Biochem.

S. Awazu and F. W. Carson,

Biophys.

Res.

unpublished

reference to the concept of allosterism is J. Monod, J. Wyman and J.-P. Changeux, J. Mo;L. Biol., g, 88 (1965). A. Krehbiel

843 (1963).

447

and M. Laskowski,

Jr.,