The effect of modifiers on the hydrolysis of esters and peptides by carboxypeptidase A

BIOCHEMICAL

Vol. 3 1, No. 4, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE EFFECT OF MODIFIERS ON THE HYDROLYSIS OF ESTERS AND PEPTIDES BY CARBOXYPEPTIDASE-A* R. C. Davies,

D. S. Auld+ and B. L. Vallee

Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School and the Division of Medical Biology, Peter Bent Brigham Hospital, Boston, Massachusetts

Received April 22, 1968

In the course of kinetic found that N-substituted and ester substrates Similar

effects

collectively

previous

activate

reports

peptidase

groups (2,10-12).

of certain

but inhibit

A we have

synthetic

esterase

available

analogous

The present

peptide

activity.

on carboxypeptidase

to

to those of such

communication

these phenomena and relates

extends our

them to the kinetic

catalysis.

and Discussion indicated,

assay employed in the present (2).

of modifiers,

the materials,

In one series of experiments

for

to those described

pre-

the effect

of a single

concentration

.05 M, was examined with 10S3 M carbobenzoxyglycyl-L-phenylalanine

concentration

L-phenylalanine

methods and conditions

study were identical

(CGP) and hippuryl-d,l-f3-phenyllactate modifier

of hydrolysis

which have properties

concerning

Unless otherwise

viously

of carboxypeptidase

have been observed with a number of compounds, referred

blocking

data currently Results

products

as modifiers,

N-terminal

investigations

was varied

(BGP) and HP&

(HPLA) '(Table I).

In another

while that of the substrate, were constant

(Figs.

* Supported by Grant-in-Aid HE-07297 frun the National of the Department of Health, Education and Welfare. + Research Fellow of the American Cancer Society.

628

series,

benzoylglycyl-

1 and 2). Institutes

of Health

BIOCHEMICAL

Vol. 31, No. 4, 1968

AND BIOPHYSICAL RESEARCH COMMUNKATIONS

TABLE I EFFECT OF MODIFIERS ON PEPTIDASE (CGP) AND ESTERASE @'LA) ACTIVITY PeptidaseFtivity(") 0

Modifier

Esterase Activity(a) %

Carbobenzoxyglycine

230

33.6

Carbobenzoxyglycylglycine

346

38

Benzoylglycine

290

32

Benzoylglycylglycine

291

33

Benzamide')

252

53.2

Benzyl alcohol

263

35

Phenol

208

47.5

Cyclohexanol

302

25

Benzene(c)

152

76

Cyclohexanone

207

44

Cinnamic acid

513

12

90

91

108

87

97

84

2,2,2-Trichloroethanol

206

30

2,2-Dimethylpropanol

260

24

3,3-Dimethylbutanol

409

14

Ethanol n-Propanol Z-Chloroethanol

(4 Peptidase

activity determined at 1 x low3 M CGP, pH 7.5, 25',-3.0 M NaCl, .02 14 Bronal. Esterase activity determined at 1 x 10 MHPLA, pH 7.5, .005 M Tris, 25'C. In each case the modifier concentration was 0.05 M.

@I .025 M benzamide. Cc)%.Ol M benzene. The hydrolysis substrate

activation

HPLA exhibits

of the peptide (2).

substrate

BGP by native

In contrast, inhibition

carboxypeptidase

the hydrolysis

(5).

of the ester analogue

A model postulating

629

displays

multiple

binding

BIOCHEMICAL

Vol. 31, No. 4, 1968

loci

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

has been proposed to account for these and other kinetic

carboxypeptidase

(10-E').

hydrolysis

of a peptide

hydrolysis

in opposite

other activity. markedly

This effect

directions,

i.e.

the hydrolysis

ought to affect activating

benzoylglycine

a product

one while inhibiting

(BG), a product both of BGP and HPLA

of CGP hydrolysis

(CG) is employed

are not restricted

to N-substituted

dipeptide

hydrolysis

They have also been observed with di- and tripeptides,

of the blocking

groups of such products

For the majority

of cqounds

is proportional

can be quite

marked.

e.g. CGG or

similar

to those

(Table I).

examined the activation

of peptidase

to the inhibition of esterase activity. Such effects -3 cinnamic acid increases Thus, at 1 x 10 M substrate,

the rate of CGP hydrolysis

five-fold

while it decreases that of HPIA eight-

(Table I). With a given modifier

activation

or inhibition

would be expected. activation.

and substrate

Thus, the hydrolysis

Hence, if a modifier

the cyclohexanol

activation

the quantitative of substrate

details

substrate

of BGP hydrolysis

(Fig.

substrate

decreases the inhibition

substrate,

concentration, 2).

inhibition,

as substrate

its effect

as demonstrated Similarly,

the

and increasing

by modifiers.

CG has also been found to activate the hydrolysis of one ester, glycolate (4) but to inhibit the hydrolysis of another, i.e. cinnamoylphenyllactate (1).

630

of

concentration,

of BGP is known to exhibit

ccanpetes with this

of HPLA is known to exhibit

HPLA concentration

pair

vary as a function

should decrease with increasing

hydrolysis

1).

by CG* had been noted

CGGG, and with a number of ccmrpounds with characteristics

*

the

(13).

products.

fold

the rate of their

of BGP, but decreases that of HPLA (Fig.

Product activation

These effects

activity

of

common to the

is even more pronounced when carbobenzoxyglycine

as the modifier. previously

and an ester pair

Indeed,

increases

This model suggests that

anomalies

o-hippuryl

for

BIOCHEMICAL

Vol. 3 1, No. 4, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-

“I a cz FVI

-0 0, 0

l.u

Figure

I I

I 2

I 3

[MODIFIER],

MX

5

I 6

lo2

1 - Activation of BGP hydrol sis (1 x low3 M) and inhibition of HPLA hydrolysis (1 x lo- 3 M) as a function of the concentration of BG (O,O> and CG (~),a). Peptidase assays were performed in 1.0 M NaCl, .05 M Tris, pH 7.5 and 2S", esterase assays in .2 M NaCl, .005 M Tris, pH 7.5 and 25O.

000

2 [CYCLOHEXAN~L~.

Figure

1 4

3

4

5

tdx102

2 - Cyclohexanol activation of EP hydrolysis at concentrations of 1 x 10-3 M (A ), 4 x 1O-3 M (m), and 2 x 1O-2 M (, ) BGP. Assays performed in 1.0 M NaCl, .05 M Tris, pH 7.5 and 25O.

631

Vol. 3 1, No. 4, 1968

BIOCHEMICAL

The chemical

and physical

properties

manner are of obvious interest. their

effects

on activation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the agents which act in this

The hydrophobic

are reminiscent

by analogous

cornpods

(3,6,7,14)

the enzyme.

Table I reveals

group of an aromatic

reciprocal

activation

and inhibition

at comparable

3,3-dimethylbutanol, analogous

fashion

gardless

of the modifiers,

the activation

hydrolysis

than straight-chain features

which in this

also

alcohols, act in

alcohols.

Re-

which may govern the effects

of BGP and inhibition

emphasize the capacity

binding

loci

of HPLA hydrolysis

of carboxypeptidase instance

differ

by

to dis-

only in the bond

locus,

bind to loci

of peptide

canpetes with the peptide

but not for the productive

the inhibition

of esterase required loci

binding,

activities

could be brought

ccxnpetition

for specific

substrate

or modifier

(product)

protein.

The extent

binding

peptide

locus.

Similarly,

if modifiers

This interpretation

were to

suggests peptide

and

through modifier

and

(10).

about directly binding

loci,

or indirectly

through

induced changes in the conformation

of confonnational

of peptide

This could

and for non-productive

as has been suggested

substrate

inhibition.

for a non-productive

peptide

for ester catalysis.

one of them is

The activation

could be understood

for ester catalysis

These effects

such results,

model (2,10-12).

can be regarded as a release

occur if a modifier

modifier

cyclohexanol

to hydrolysis.

based on the multiple

overlapping

either;

of the blocking

The bulky aliphatic

While a number of mechanisms could explain

binding

also produce

and 2,2,2-trichloroethanol

and physical

between substrates,

susceptible

changes.

with

for such

The aromaticity

requirement

and are more effective

the same agent further criminate

acid is not required

2,2-dimethylpropanol

of the chemical

interactions

of these agents have hydrophobic

concentrations.

produces very marked and similar

and

of chymotrypsin

since amides and alcohols

does not seem to be a specific

groups

of the inhibition

that while all

a carboxyl

effects

of the modifiers

and suggest hydrophobic

character,

similar

nature

of the

change could depend on the exact

632

vol. 31, No. 4, 1968

structural

features

productive

binding

products

BlOCHEMlCAL

of substrates and catalysis.

and other modifiers

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

or modifiers

and could further

Such a postulate

would seem reasonable

effect

both

for the mode of action since conformational

changes

of the enzyme have been observed in the crystalline

state

on binding

of at

least

(8,9).

Studies

of the

one peptide

effects

of modifiers

and peptides

substrate,

i.e.

glycyl-L-tyrosine

on the carboxypeptidase

are presently

catalyzed

hydrolysis

of

of esters

being extended to examine these hypotheses. References

1. 2. 3. 4. 2: 7. 8. 9. 10. 11. 12. 13. 14.

Awazu, S., Carson, F. W., Hall, P. I,., and Kaiser, E. T., J. Am. Chem. Sot. g, 3627 (1967). Davies, R. C., Riordan, J. F., Auld, D. S. andvallee, B. L., Biochemistry S-, 1090 (1968). Hymes, A. J., Robinson, D. A. and Canady, W. J., J. Biol. Chesn. 240, 134 (1965). Kaiser, E. T., Awazu, S. and Carson, F. W., Biochem. Biophys. Res. Cmun. 2l, 444 (1965). McClure, W. O., Neurath, H. and Walsh, K. A., Biochemistry 3, 1897 (1964). Miles, J. L., Robinson, D. A., and Canady, W. J., J. Biol. &m. 238, 2932 (1963). R~PP, J. R., Niemann, C. and Hein, G., Biochemistry 5, 4100 (1966). Reeke, G. N., Hartsuck, J. A,, Ludwig, M. L., Quiocho, F. A., Steitz, T. A. and Lipsccanb, W. N., Proc. Natl. Acad. Sci. U.S. 5& 2220 (1967). Steitz, T. A., Ludwig, M. L., Quiocho, F. A. and Lipscanb, W. N., J. Biol. Chm. 242, 466 (1967). Vallee, B. L., 152ndMeeting Am. Chem. Sot. 83C (1966). Vallee, B. L., Abstract VIIth Internatl. Congr. Biochem. (Tokyo) 194.(1967). the Vallee, B. L., Symposim on the Electronic Aspects of Biochemistrjjf N. Y. Acad. Sci. (Dec., 1967), (in press). Whitaker, J. F., Menger, F., and Bender, M. L., Biochemistry 5, 386 (1966). Wildnauer, R. and Canady, W. J., Biochemistry 5, 2885 (1966).