Isolation of a specific acyl-chymotrypsin intermediate

Vol. 24, No. 6, 1966

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ISOLATION OF A SPECIFIC ACYMXYMOTRYPSIN

INTERMEDIATE

Y. Shalitin Department

of Chemistry,

Israel

Institute

of Technology,

Haifa,

Israel

J. R. Brown* Department

of Biophysics,

ReceivedJuly It

is generally

involves

a three

complex,

formation to free data,

occurring

phenyl

acetate

Enzymes"

step mechanism:

on catalyzed during

to the active

direct

evidence

of chymotrypsin

Israel

hydrolysis

by reacting

lo3 or lo4 fold

faster

study an acyl-enzyme after

prior

-chymotrypsin

derivative

voltage

paper

electrophoresis,

peptide

which

contained Fellow

in acid

soiution.

pepsin

of U.S.

with

specific

After

and separation

Public

serine IIealth

817

p-nitroAcetyl-

pnitrophenyl group

1958).

was

Such

substrates,

which

acetate. by rapid

the chromogenic

the nitrotyrosyl

the active

1965).

the acetyl

compound was prepared with

with

and Kezdy,

p-nitrophenyl

incubation

ester

of compounds like

and van Andrichem,

than

(by hydro-

or on spectrophotometric

was sholm that

(Oosterbaan

and

is based mainly

the enzyme with

has not yet been demonstrated

ethyl

Postdoctoral

(Bender

and it

and deacylation

enzyme hypothesis

the enzymatic imidaeole

of esterolytic

of an enzyme-substrate

intermediate,

reactions

-3-nitrotyrosine

l

Rehovoth,

acetylcholineesterase)

establishment

transfer

serine

pathway

trypsin,

The acyl

and Wood, 1956)

linked

In this

rapid

of an acyl-enzyme

has been prepared

(Balls

the catalytic

(chymotrypsin,

and cinnemoyl

-chymotrypsin

are hydrolyzed

that

enzyme and acid.

shifts

acetate

accepted

"serine

on kinetic

of Science,

22, 1966

proteolytic

lysis)

The Weizmsnn Institute

denaturation

substrate

digestion

H-acetyl-

of the acyl-

of the peptides

by high

group was found attached

residue. Service,

G!+16,

577.

to a

Vol. 24, No. 6, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Preparation of N-acetyl-3-nitrotyrosyl-chvmotrvosin Chymotrypsin (Worthington 3 x crystallized,

20 mg) was dissolved in

HCl (0.001 M, 10 ml) and incubated at 20' with N-acetyl-3-nitrotyrosine

ethyl

ester (3 mg) and after 5 minutes an equal volume of 10 percent ttichloroacetic acid was added. The precipitated

protein was centrifuged and washedthree

times by resuspension in 5 percent trichloroacetic of the nitrotyrosine

derivative

A part of the precipitate

acid, after which no traces

could be detected in the supernatant solution.

was dissolved in sodiumhydroxide, 0.1 N, and from

the absorbsncy due to protein at 280 mu and that due to the nitrophenolate chromophoreat 420 mu it was calculated that 0.5 nitrotyrosyl

groups were

bound per protein molecule. Isolation

of a nitrotyrosvl-nentide

A pepsin bydrolyzate of nitrotyrosyl-cbymotrypsin derivative,

(10 mg protein

1 mg pepsin, 2 ml 5 percent fonzic acid, 37', 16 hours) was

applied as a 20 cmband on WhatmanNO. 1

paper

and the peptides were sepa-

rated by high voltage electrophoresis (60 volts per cm, 40 min, 25') using pH 6.5 buffer (pyridine acid: water: 25:1:25 v/v)

and xylene as coolant.

The nitrophenolate chromophorewas easily detected by exposing the dried electrophoretogram to ammoniavapour. Two yellow zones were thus observed, one cationic (2.2 cm from neutral) and one anionic (15.1 cm from neutral).

The mobility

N-acetyl-3-nitrotyrosine

of

the anionic yellow zone was the sameas that of

run as marker on the samepaper. Also, the anionic

yellow zone did not coincide with any coloured zone on a guide strip developed with Cd-ninhydrin reagent, indicating to a peptide.

that the nitrotyrosyl

group is not linked

The cationic yellow band coincided with a zone that gave a brown

colour with Cd-ninhydrin reagent and fluoresced under a U.V. lsmp. This zone

818

Vol. 24, No. 6, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was eluted from the paper, the dried eulate hydrolyzed (6 N HCl, llO", 24 hours), and the hydrolyzate analyzed in a Beckmann-SpincoAmino Acid Analyzer, Free 3-nitrotyrosine

was also run in the amino acid analyzer and it

appeared 12 minutes after the phenylalanine peek (50 cm column, 70 ml per hour).

HESULTS An initial

attempt to trap the nitrotyrosyl

pH 7 by denaturation wasunsuccessful. nitmtyrosyl

enzyme intermediate at

However, at pH 2.5 a denatured stable

chymotrypsin could be prepared in about 50 Per cent yield.

Identification

of the locus of attachment of the nitrotyrosyl

residue

to the enzymewas based on the finding by Brown and Hartley (1966) that a double cystine peptide containing the active center serine can be easily detected and purified by high voltage electrophoresis at PH 6.5 of a pepsin digest of chymotrypsin.

We therefore digested the denatured nitrotyrosyl

-

-chymotrypsin with pepsin and separated the peptides under the conditions reported by Brown and Hartley (1966) and indeed on exposure of the paper electrophoretogrem to auunoniavapour, the nitrotyrosyl-group

was detected as

a yellow zone which otherwise appeared the sameas the active center peptide (e.g. electrophoretic

mobility,

ninhydrin colour, fluorescence under a U.V.

lamp). It should be pointed out that becauseof its large size for a single positive charge, the active center peptide is well resolved from all other peptides in the one dimensional fingerprint of the nitrotyxvsyl-peptide

of pH 6.5.

is shownin Fig. I.

The proposedstructure

It was deduced from the

amino acid composition (Table 1) of the peptide and the amino acid sequence of chymotrypsin (Hartley,

1964), and pairing of disulfide bonds reported by

Brown and Hartley (1963).

The observed amino acid composition is in good

agreementwith that expected for the structure in Fig. 1.

819

The somewhatlOW

Vol. 24, No. 6, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE

1

The amino acid composition of a nitrotyrosyl-peptide isolated for a pepsin digest of nitrotyrosyl-chymotrypsin and the amino acid composition expected for the active serine peptide structure given in Fig. 1.

Composition

Amine Acid

Compcsition

Amino Acid

Observed Expected

Observed Expected

Aspartic acid

2.4

2

Isoleucine

0.0

0

Threonine

6.7

7

Leucine

1.5

2

Serine

6.2

6

Tyrosine

0.8

1

Glutamic acid

0.1

0

Phenylalanine

0.3

1

Pmline

2.0

2

Nitrotyrosine

0.3

1

Glycine

8.6

9

TrJrptophan

+

2

Alanine

2,8

3

Lysine

1.9

2

Half-cystine

3.5

4

Histisine

0.0

0

Valine

3.5

3

Arginine

0.0

0

Methionine

1.0

1

P P Phe-Ala-Ala-Gly-JTMhWCyS-Val-ThMhtilyary-Gly-Leu 130 \

143

Acetyl-nitrotyr-0 1 Se~et-Cly-~ply-Gly-P~-~u-Val~yS-Lys-Lys-~~ly-Ala~~-~ 190 \ 195

208

Fig. 1. Proposed structure of N-acetyl-3-nitrotyrosyl-peptide based on the sequenceand numberinggiven by Hartley (1964) and the S-S pairing according to Brown and Hartley (1963, 1966). P designates partial splitting by pepsin.

820

Vol. 24, No. 6, 1966

BIOCHEMICAL

values of threchine, glycine, due to partial

splitting

and valine are difficult Hartley (1966).

leucine, tyrosine and phenylanine are probably

by pepsin. The somewhathigh values of aspartic acid to explain, but these were also observed by Brcwn and

Half-cystine

low recovery of nitrotyrosine tryBin

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is partially

destroyed by acid hydrolysis.

can be ascribed to partial

The

labeling of chymo-

and to hydrolysis during the pepsin digestion, since free acetyl-

-nitrotyrosine

was also fomd in the fingerprint

There is therefore little

of the pepsin hydrolyzate.

doubt that the nitrotyrosyl

group is linked to the

active center peptide, probably on the active serine as indicated in Fig. 1. As there are manyother serihe and threonine residues which could be sites of acylation,

it would be desirable to degrade the peptide to smaller

fragmenta so as to have unambiguousproof for the site of attachment. attempt to do this by digestion with subtilisin

An

failed becausethe nitretyro-

syl group was also completely removed as N-acetyl-nitrotyrosine. searching for ways to degrade the nitrotyrosyl-peptide

Ne are

which nil1 leave the

chromophoreattached. DISCUSSION A stable acetyl-chymotrypsin has been isolated, after treating the enzymewith p-nitmphenyl acetate at pH 5 (Balls and Wood, 1956). By aubjetting

this acetyl-enzyme to proteolytic

thee (1958) were able to identify

digestion Oosterbaan and van Andri-

the site of acylation as serine 195.

Since specific substrates of chymotrypsin, like N-acetyl tgrosine ethyl ester or N-acetyl-3-nitrotyroeina

ethyl ester (a somewhatbetter substrate),

are normal estera but nevertheless are hydrolyzed 103 -104 fold faster than active esters such as p-nitrophenyl acetate or cinnamoyl imidazole, it was reasonable to question whether the samecatalytic

pathway exists in each case.

Several lines of evidence based on kinetics or spectral changesof the enzyme reaction uith specific substrates strongly support the acyl enzymehypothesis.

821

Vol. 24, No. 6, 1966

BIOCHEMICAL

For instance, with

when a series

chymotrypsin

alcohol

deacylation

Incubation

transfer

alcohol,

reactions

where

controlling

(Bernhard

1964) and 0

18

with

its

reacted

and HP0

(Bender

18

acids

(Sprinson

was formed,

the

1964).

in the presence gave rise

and Glasson,

1960;

of the

Bond and Bender,

substrates

et al.,

labeled

intermediate

(Zerner,

hydroxylamine esters

esters

was the same, irrespective

a co:mon acyl

of chymotrypsin

1864) hydroxamates et al.,

that

was rate

like

or phenylalanine

of hydrolgsis

suggesting

of which

nucleophiles

of tryptophane

the rate

moiety,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1960;

of

to catalyzed Bender -*et al

Epand and Wilson,

1963;

f

Bender

and Rittenberg,

1951)

were

when a specific

substrate

chymotrypsin,

a biphasic

formed. It has been demonstrated N-aoetyl-L-tryptophan spectral

ethyl

ester

change took place

mechanism

(Kezdy,

In this

Clement

recently

which

reacted

with

is consistent

and Bender,

work we tried

that

with

1964).

to find

direct

enzyme, by isolating

it.

evidence

the specific

acyl

nitrotyrosyl

enzyme intermediate

by denaturation

chymotrypsin

hydrolyzes

tyrosine

this

@I (about

second)

place.

200 substrates

and because

the trapping

-nitrotyrosyl

molecules

of the relatively

experiment

However,

intermediate

N-acetyl

acid

compound should

the existence to trap

at neutral

ester

substrates

are cleaved

be in the order

Since

very rapidly

at per

of enzyme needed denaturation

the lifetime

of a minute

by denaturation

the N-acetyl

by an enzyme molecule

concentration

(pH 2-3)

of

pH failed.

was consumed before

conditions

enzyme was trapped

for

Our attempt

high

the substrate

under

acylation-deacylation

for

took

of the acyl

and thus the acetyl-

and identified

by its

yellow

colour. The results -enzyme intermediate been demonstrated However,

presented

in this

is formed with under

as the @I profile

conditions

paper

give direct

a good substrate, well

below

of the chymotrypsin

822

that

evidence although

that this

an acylhas only

of optimum activity.

reaction

rate

shows that

from

Vol. 24, No. 6, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

pH 2 to 8 the rate is dependent on a single basic group of pC 6.8 it is reasonable that the aamemechanismoperatea at optimal pH aa in lower pH values. The exact site of acylation on the enzymehas not yet been determined, due to the lability fragmentation. end identify

of the nitrotyroayl-peptide

towards further

Attempts are being directed to overcomethese difficulties

the site of acylation.

But as the nitrotyrosyl

attached to a peptide containing the 'active that this is the site of the tyrosyl

group ie

serine 195" it is probable

linkage.

We thank Prof. Ephraim Katchal&.i for helpful diacusaionz,

REFEXENCES Balls, A.K., and Wood, H.N. (1956). J. Biol. Chem.a, 245. Bender, ML., Clement, G.E., Gunter, C.R., and Kezdy, F.J. (1964). J. Am. Chem.Sot. &, 3697. Bender, M.L. and Glaseon, W.A. (1960). J. Am. Chem.SOC., 82, 5336. Bender, ML. and Kezdy, F.J. (1965). Ann. Rev. Biochem., 2, 49. Bernhard, %A., Coles, W.C. and Nowell, J.F. (1960). J. Am. Chem.Sot., 82, 3043. Brown, J.R. and Hartley, B.S. (1963). Biocham. J., 3, 59P. Brown, J.R. and Hartley, B.S. (1966). In press. Epand, R.N. and Wilson, I.B. (1963). J. Biol. Chem., a, 1718. Hartley, B.S. (1964). Nature, 201, 1284. Kezdy, P.J., Clement, G.E. and Bender, M.L. (1964). J. Am. Chem.Sot., 86, 3690. Ooaterbaen, R.A. and van Andrichem, M.E. (1958). Biochem. Biophys. Acta, 2, 423. Sprineon, D.B. and Rittenberg, D. (1951). Nature, 167, 484. zerner, Be, Bond, R.P.M. and Bender, N.L. (1964). 3. Am. Chem.Soo. s, 3674.

823