Stereospecific activation of chymotrypsin catalyzed hydrolyses

BIOCHEMICAL

Vol. 25, No. 1, 1966

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

STERECSPECIFIC ACTIVATION OF CHYMOTRYPSIN CATALYZEDHYDROLYSES* DagmarR. Ponzi and George E. Hein** Department of Chemistry, Boston University,

Received August

23, 1966

We wish to report

that the optical

methylpropyl)-trimethylammonium I-

Boston, Mass., O22l.5

antipodes of (l-carbomethoxy-2-

iodide,

[(CH3)2CHCH2CH(C02CH3)N(CH3)3]'

(CaMP) are modifiers of chymotrypsin catalyzed

ety of substrates, an activator

hydrolyses of a vari-

and that the L-isomer, but not the D-isomer acts as

of the hydrolysis

ester and of the hydrolysis

of both benzoyl-D- and L-alanine methyl

of No-bensoyl-L-arginine

methyl ester.

type of behavior observed for both modifiers depends dramatically substrate.

The enzyme displays distinct

stereospecificity

two isomers of the quaternary ammoniumsalt:

significant

The on the

towards the activation

is

only observed with the L-antipode. In general, it has been assumedthat the enzyme chymotrypsin possesses but a single binding site.

This belief

deal of evidence ranging from equilibrium Briggs, 1952) to a kinetic zyme catalyzed

hydrolysis

pairs of inhibitors

dialysis

of a typical

(Rapp, 1964).

and similar

studies (Loewus and

analysis of the observed velocity

of the en-

substrate in the presence of

Studies in which reversible

tors "protect 'I the enzyme from denaturation, inhibitors

is supported by a great

inhibi-

the action of irreversible

phenomenacan also be cited to support the view

that chymotrypsin has but a single binding cavity. *

We gratefully acknowledge the support of this investigation by the Public Health Service (Grant GM1011-03) and the National Science Foundation (Grant GB-795).

**

Department of Biological Mass., 02115.

Chemistry, Harvard Medical School, Boston,

60

Vol. 25, No. 1, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Yet, a certain amount of evidence suggests that chymotrypsin can, under certain molecules.

circumstances, accommodateat least

Original

two small organic

reports concerning activation

by excess substrate

(Wolff and Niemann, 1959; Wolff and Niemann, 1963) (Trowbridge, et al.,,19633 (Wallace, et al., tivation

Ingles and Knowles, 1965)

methyl ester (Trowbridge, et al.,

of the hydrolysis

and defended

Less ambiguous is the reported substrate ac-

of the chymotrypsin catalyzed hydrolysis

L-arginine

et al.,

1964).

have been questioned

of bifunctional

1963)

of No-toluenesulfonyland the activation

substrates by 9-aminoacridine (Wallace,

1966).

Whenone considers that the natural substrates of chymotrypsin are polypeptides, interacting

it would not be surprising

if the enzyme were capable of

with a multi-amino acid segment of such a chain, and there

is scme indirect

evidence to support this view (Neil,

et al.,

Translated into terms of small substrates or modifiers,

it

1966).

is at least

reasonable to suggest that the enzyme can bind two or more of these simultaneously. MATEKULSANDMETHODS Substrates were prepared in the usual manner by acylation corresponding amino acids followed by esterification 1958).

Kinetic

kinetic

(Applewhite,

studies were carried out on a p&stat

has been described in the literature

(Applewhite,

of the et al.,

in the manner which

et al.,

1958).

The

data were analyzed by the method of Boomanand Niemann (1956)

with the aid of an IBM 1620 digital cribed by Abrash, et al.,

(1960).

computer and a program written

as des-

Modifiers were prepared from the cor-

responding D- and L-amino acids by esterification

with thionyl

in methanol (Brenner end Huber, 1953): The L-ester hydrochloride 0.29 mole) was dissolved in 300 ml. of dry acetonitrile

chloride (53 g.,

and refluxed

in a

dry system containing 86 g., 0.60 moles of potassium carbonate and 186 g., 1.31 moles of methyl iodide for 60 hours.

61

Additional

amounts of methyl

Vol. 25, No. 1, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

iodide (40 ml., 0.60 moles each time) were added at 12 hour intervals. The mixture was filtered

and the solution evaporated to leave a crude

product which was crystallized

three times from methanol.

pure quaternsry salt was 49 g- (54%). m.p. 187-189 (2, % H20).

w.

Calcd: for C10H2g021 (315):

I, 40.3; Found: C, 38.0; H, 6.8; I, 40.3. similarly

in 36%yield,

dec.,

The yield [a],

of

+ 12.00

C, 38.1; H, 7.1;

The D-isomer was prepared

m.p. 185-187 dec., [a],

- 11.8O (2, 2% H20).

Found: C, 37.7; H, 7.1; I, 40.2.

Anal.

In the experiments reported here, substrate concentration6 proximately

were ap-

equal to Kc values observed for these compoundsin our sys-

tem (0.5 M KI). 7.9 and total

All reactions were carried out at 25.0 2 0.1, at pIi ionic strength 0.5 M consisting of modifier and sodium

iodide. BESDLTSANDDISCUSSION Evidence that both the quaternery ammoniumcompoundsinteract

with

the enzyme could be obtained from the results with methyl hippurate. Both isomers proved to be inhibitors range studied. tration,

of the reaction in the concentration

The degree of inhibition

varied with substrate concen-

but did not follow any simple inhibition

ative data are shown in Fig. 3.

The effect

type.

The represent-

can be attributed

neither

to iodide ion nor to quaternary groups in general since the concentration of iodide ion was 0.5 molar in all terminations

experiments and independent de-

showed that tetramethylammonium iodide had no effect

reactions studied up to the limit ionic strength.

The hydrolysis

of its of neither

on the

solubility

CO.25 M) at constant

optical

antipode of Cal@ was

catalyzed by the enzyme. Despite the evidence that both modifiers reversibly the

enzyme,

only the L-antipode showedsignificant

rate of hydrolysis cases activation

of benzoyl-L-

interact

modification

and D-a&mine methyl esters.

with of the

In both

was observed, and for the L- ccnnpounda maximumwas

62

BIOCHEMICAL

Vol. 25, No. 1, 1966

AND BIOPHYSICAL RESEARCH COMMUNiCATlONS

ftx - g - Ala-OCH,

8x-&-

1.e -

Ala-OCHx

i

1.6 .4 +

14 -.

Cow.

d

+

Modiilrr

1 and 2. Effect of L- and D- CaMPon the chymotrypsin catalyzed hydrolyses of benzoyl-D-alanine methyl ester and benAll reactions at 25.0°, pH 7.9, zoyl-L-alanine methyl ester. total ionic strength 0.5 M by addition of sodium iodide. Fig. 1: l L-CaMP, CSI = 2.59 X 10a3M; 0 D-CaMP[Sl = 2.59 X 10m3M. Fi 2: 0 L-CaMP, CSI = 0.912 x 10-3~; I) L-cde, Csl = 4.44 X 10' TM; o D-CaMP, Csl = 2.38 X 10-3M.

Figures

No-

Br-

I-

Arg-OCH,

Mathyl

Hippuratr

1.4

1.2 f: “” -

LO

“0 0.t l-.

+! t,g 2. --#---..- _ - -_-s---4- - #---0 0

-__

-----------

--_------------_

8 I

0.1 6--

Q 0.4 I -.

4

Oi ! -. I

I I

I I

I

I

0.1

0.2

0.3

04

0.5

M

of

Modillrr

Cont.

4

I I

I

I 1

I 1

I I

0.1

0.2

0.3

0.4

03

M

Figures 3 and 4. Effect of Ir and D-CaMPon the chymotrypsin catalyzed hydrolyses of p-benzoyl-Garginine methyl ester and All reactions at 25.0°, pH 7.9, total ionic methyl hippurate. Fig. 3: 0 L-CaMP, strength 0.5M by addition of sodium iodide. Fig. 4: l L-CaMP, CSI = 14.6 X KY3M; 0 D-CaMP, 12.3 X 10e3M. [Sl = 1.91 X 10-3M; o D-w¶P, Csl 2 2.36 x 10"M.

63

Vol. 25, No. 1, 1966

BIOCHEMICAL

noted at approximately

0.2 M.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(Fig.

1 and 2).

As might be expected, the degree of activation on the concentration

of substrate and of modifier.

was dependent both Finally,

similar

results were obtained when the two compoundswere tested as modifiers of the chymotrypsin catalyzed ester.

A qualitative

hydrolysis

of fl-benzoyl-L-arginine

methyl

summaryof the results is presented in Table I. Table I

Behavior of D- and L-(l-carbomethoxy-2-methylpropyl)-trimethylammonium iodide as modifiers of the chymotrypsin catalyzed

hydrolyses of acylated

amino acid esters.a Effect

of smmoniumsalt

Substrate

L-isomer

D-isomer

methyl hippurate

mixed inhibition

mixed inhibition

Benzoyl-L-alanine

methyl ester

activation

slight

inhibition

Denzoyl-D-alanine

methyl ester

activation

slight

inhibition

ff-Be nzoyl-L-arginine a

methyl ester activation

All reactions at 25.0% pl? 7.9, total sisting of modifier and NaI.

inhibition ionic strength 0.5 M,con-

The substrate dependent behavior of L-CaW and the different

type

of behavior of its antipode, D-CaMF',adds a new dimension to the study of chymotrypsin specificity. activity

The observation of activation

with somecompoundsis prima facie evidence for the formation

of a ternary

enzyme-substrate-modifier

complex.

cupied by the modifier shows stereospecificity structural hibit

of catalytic

specificity

The secondary site ocand also at least some

since other organic molecules, e.g.,

indole,

the enzyme catalyzed hydrolyses of the alanine derivatives

in-

(Hein

and Niemann, 1962). The type of explanation for trypsin

suggested by Inagami and Murachi (1964)

does not appear to be applicable , since the alenine deriva-

64

BIOCHEMICAL

Vol. 25, No. 1, 1966

tives and No-benzoyl-L-arginine Yrifunctional"

substrates.

is observed with better have a slight

inhibitory

acetyl-lvaline

RESEARCH COMMUNICATIONS

methyl ester must be considered as It may be significant

chymotrypsin substrates. effect

that no activation Both L- and D-CeMP

on the enzyme catalyzed

and -phenylalanine

It may also be significant a&nine

AND BIOPHYSICAL

hydrolyses of

methyl esters.

that L-CaMP, fl-toluenesulfonyl-L-

methyl ester and 9-aminoacridine , the compoundswhich activate

chymotrypsin towards somesubstrates , all possess a positive It is conceivable that chymotryprin binding site,

end even that this site plays a functional

secondary specificity et al.,

contains a "secondary",

of the enzyme towards polypeptide

anionic

role in the substrates (Neil,

However, such speculations must await further

1966).

charge.

experiments,

possibly using suitable enzyme modifiers. REFERENCES Abrash, Ii. I.,

Kurtz,

A. N. and Niemann, C., Biochem. Biophys. Acta, s,

378 (1960).

Applewhite, T. H., Waite, H., and Niemann, C., J. Am. Chem. Sot., &,

1465 (1958). Booman, K. A. and Niemann, C., J. Am. Chem. Sot., 78, 3642 (1956). Brenner, M. and Huber, W., Helv. Chem. Acta, &, 1109 (1953). Hein, G. E. and Niemann, C., J. Am. Chem. Sot., 84, 4487 (1962). Inagemi, T. and Murachi, T., J. Biol. Chem., 2 9, 1395 (1964). Ingles, D. and Knowles, J. R., Biochem. J., 2,8 719 (1965). Loewus, M. W., and Briggs, D. R., J. Biol. Chem., 199, 857 (1952). Neil, G., Neimann, C. and Hein, G. E., Nature, 210, 903 (1966). of Technology, 1964. R~PP, J. R-3 Ph.D. Thesis, California Institute Trowbridge, C. G., Krehbiel, A. and Laskowski, M., Biochemistry, 2,

843 (1963).

Wallace, R. A., Peterson, R. L., Niemann, C. and Hein, G. E., Biochem. Biophys. Res. Comm.,3, 246 (1966). Wolff, J. P. III, and Niemann, C., Biochemistry, 2, 82 (1963). Wolff, J. P. 111, and Niemann, C., J. Am. Chem.Sot., 81, 1012 (1959). Wolff, J. P. III, Wallace, R. A., Peterson, R. L. and Kernann, C., Biochemistry, 1, 940 (1964).

65