Kinetic evidence for an acyl-enzyme intermediate in the α-chymotrypsin-catalyzed hydrolysis of N-acetyl-L-tryptophan ethyl ester

BlOCHEMlCAL

Vd. 12, No. 4,1963

AND BlOPHYSICAl

RESEARCH COMMJNICAllONS

KINETIC EVIDENCE FOR AN ACYL-ENZYME INTERMEDIATE IN THJ3 a-CHYMOTRYPSIN-CATALYZEDHYDROLYSIS OF N-ACETYL-L-TRYPTOPHAN ETHYL ESTER* Myron L. Render++ and Gerald E. Clement*** Department

of Chemistry,

Northwestern

Evanston,

In a recent the methyl, L-tryptophan

and p-nitrophenyl

with

step process,

for

be hydrolyzed in rates.

plained

if

(ES')

rate

constant

1963).

This

in terms of a onereactions,

to which

would demand that

the three

at a hundred-fold This behavior

one postulates

intermediate

of N-acetylat pH 7.0 by

and Render,

nucleophilic

conforms,

ference

position

(Zerner

cannot be rationalized

chymotrypsin

laboratory,

esters

the same catalytic

(maximal velocity).

esters

from this

were shown to be hydrolyzed

a-chymotrypsin behavior

Illinois

communication

ethyl

University

or greater

can, however,

the formation

(equation

be ex-

of a common

1) whose subsequent

(k3) is rate-determining.

dif-

decom-

For these three

Whis research was supported by grants from the National Institute of Health. Paper XXIII in the series, the mechanism of action of proteolytic enzymes. **Alfred P. Sloan Foundation Research Fellow. ***National Institutes of Health Postdoctoral Research Fellow. 339

vol. 12, No. 4,1963

BIOCHEMICAL

substrates,

AND BIOPHYSICAL

tne common intermediate

RESEARCH COMMUNlCAllONS

is N-acetyl-L-trypto-

phanyl-a-chymotrypsin. E+S

&ES

---+ES'A> +

E+Ps

(1)

PI

tion

In a second communication,

a mechanistic

was presented

the pH-rate

the hydrolysis determining

showing that

of those substrates is a bell-shaped

k3 is rate-determining 1963).

If

pH-rate

profile

while

file

curve while

(Bender,

a bell-shaped

k3 shows a sigmoid pH-rate of N-acetyl-L-tryptopha

it would be expected

of

those in which

curve.

indeed k2 exhibits

in the hydrolysis

ester,

profile

in which kz is rate-

is a sigmoid

et al.,

correla-

proethyl

that the rate-determining

step which was shown to be k3 at pH 7 would change to kz at some high pH, for "leg"

of the bell

sigmoid etic lysis

curve.

constants

at some high pH the right-hand

must cross below the "flat" We have therefore

for

determined

of the the kin-

the a-chymotrypsin-catalyzed

of N-acetyl-L-tryptophan

ethyl

ester

hydroover a wide

range of pH from 2.5 to 11.6.* Equation and 3 (Gutfreund

1 has been shown to lead to equations and Sturtevant,

and sigmoid pH-rate lation

profiles

1956).

of acylation

The bell-shaped (k2) and deacy-

(k3) can be analyzed in terms of equations

5 respectively.

Using these equations

2

together

4 and with

*These experiments were possible since it was shown that a-chymotrypsin was stable over the entire pH range during the course of kinetic measurements. Denaturation studies were carried out by titration with N-cinnamoylimidazole. 340

the

Vol.

12, No. 4, 1963

BIOCHEMICAL

AND BIOPHYSICAL

r,((apPl

= Q/(ka

kcat

= k2k3/(k2

k2

= k2(lim)/(l

+ (H+)/Kl

k3

= k3(lim)/(l

+ (H+)/(K,~)

conclusions

+ $1

of the previous

trypsin-catalyzed

hydrolysis follow

(3)

a sigmoid

kcat will

decrease

(5) the follow-

curve to some high but above some pH,

(as k 2 becomes rate-determining);

and (2) Km(app) will

increase

(as the rate-determin-

ing step changes from k3 to ka), value

leading

eventually

which is the true K,.

iments shown in Fig.

The exper-

1 bear out these predictions.

1, the change in rate-determining

step

from k3 to kz may be seen both in curve B, kcat, changes from a sigmoid

curve to a bell-shaped

about pH 10, and in curve C, K,(app) about the same pH from its its high value. &a,t&,(app> it

(curve

Fig.

A) is a true from equations

which changes at value

systems is pH independent,

kcat&(app)

bell-shaped

curve as

2 and 3 this

The circles culated

quotient data on

the pH dependence of

= kp/Km is simply the pH dependence of

k2, a bell-shaped The solid

to

1 shows that

is equal to k2/Km; since Km from independent other

which

curve at

low pH-independent

Furthermore,

should be, for

(4)

of N-acetyl-L-tryptophan

k3 is rate-determining),

In Fig.

+ K;(/(H+))

(1) kcat of the a-chymo-

pH (while

to the limiting

(2)

communications,

can be made:

ester will

Km

+ kj)

ing predictions ethyl

RESEARCH COMMUNICATIONS

curves

curve. of Fig. of Fig.

in the following

be combined and rearranged

1 are experimental

1 are theoretical way. to 341

Equations

points.

curves cal2 and 3 can

Vd. 12, No. 4,1963

BIOCHEMICAL

5

6

7

AND BIOPHYSICAL

6

9

IO

II

RESEARCH COMMUNICATIONS

12

PH

Fig.

1.

The a-chymotrypsin-catalyzed N-acetyl-L-tryptophan ethyl in 0.81% acetonitrile-water: B, &at; 0, %(app).

l/Km(app) At pH values therefore,

= l/Km + (k,at/Km(app))(l/k3)

greater

than 8, k3 = kj(lim),

above pH 8 a plot

should be linear cept l/Km.

hydrolysis of ester at 25.00 A, kcat&(app);

with

of l/Km(app)

a constant; vs. k,.t/Km(app)

a slope of l/kj(lim)

The expected linearity

(6)

and an inter-

was observed

yield-

ing a Km of 4.1 x 10'3M for N-acetyl-L-tryptophan ester.

Knowing Km, one may calculate

kz(lim)

(corresponding

to the right

eqs. 2 and 4.

pK1 and pK,( can be calculated

from plots sults

leg of the bell)

and pKs from

of kcat]Km and kcat respectively.

of these calculations

The theoretical

curves

are shown in Table

of Fig.

the data of Table I, together tion

ethyl

of equations

2-5.

1 were calculated with

the proper

directly The reI. using combina-

It is seen that the agreement 342

Vol. 12, No. 4, 1963

BIOCHEMICAL

between experiment ting

that

AND BIOPHYSICAL

and theory

the bell-shaped

the acylation

is quite

RESEARCH COMMUNICATIONS

good, indica-

and sigmoid dependencies

(ka) and deacylation

(k3) reactions

for are

Table I The a-Chymotrypsin-catalyzed

Hydrolysis

tryptophan Step

of N-acetyl-E

Ethyl Estera

k(lim)

kcalcd. c @,C sec." sec.-'

pKa

sec.-' Acylation(ks)

1818

Deacylation(k3)

6.77,

46.5

9.21

1100

480

27

29

6.86

a. The kinetics were followed spectrophotometrically at 300 q measuring initial rates ((Eo)<((So)). The exact experimental techniques have been described by Bender et al. (1962). b. Calculated by Zerner and Bender (1963) by an independent experimental approach. c. pH 7.0. verified action

and further (equation

that

the stepwise

nature

of the re-

1) and the change in rate-determining

step of reaction

with pH are verified.

The agreement

between the rate

constants

and deacylation

determined

in the present

agreement with entirely

of acylation experiments

those calculated

different

kinetic

In the low pH region tional

sigmoid

previously

of 2.5 to 5.04,

I).

an addi-

dependence of kcat may be observed,

the kinetic

importance

apparent

pKa of 4.

The apparent

(280 times)

from an

argument (see Table

implying

corresponding

are in reasonable

to this

part

than the limiting

of a base with limiting

of the reaction rate

constant

rate

an constant

is much less at pH 7.9.

Vol.

12, No. 4,1963

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNlCATlONS

A pH dependence of Km, corresponding

to this,

observed

1962; Stewart

previously

et al.,

1963).

interpretation snetic

(Bender,

A detailed

et al., report

and mechanistic

pH dependence will

has been

of the theoretical

implications

be the subject

of this of a future

publication. The data for

the a-chymotrypsin-catalyzed

sis of N-acetyl-L-tryptophan

ethyl

to conform to the predictions equation

1, involving

general action

the overall will

consist

as one extreme, and with

ester

are thus seen

of the two-step

an acyl-enzyme

hydrolymechanism,

intermediate.

In

pH dependence of a chymotrypsin of a family

of curves,

with

a sigmoid dependency at the other

re-

curve A extreme,

curve B as only one of number of intermediate

examples. EHENCES Bender, M. L., Clement, G. E., Kezdy F. J. and Zerner, B J. Am. Chem. Sot., &T, 357 (19633. BgAder, M. L., Schonbaum, G. R. and Zerner, B., J. Am. Chem. Sot., &, 2562 (1962). Clement, G. E. and Bender, M. L., Biochem., 2, 0000 Gutfreund H. and Sturtevant, J. M., Biochem. ?96&* 656 (1956): &WE& J. A., Le, H. S. and Dobson, J. E., J. Am. Chem. Sk., 82, 1537 (1963). Zerner, B. and Bender, M. L., J. Am. Chem. Sot., &, 356

(1963).

344