Vol. 66, No. 3, 1975

AMINO

BIOCHEMKAL

ACID

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SEQUENCE AROUND A ALCOHOL

Ciro Istituto poli,

Via

Received

Colonna

Biologica,

Costantinopoli

August

CYSTEINE

OF YEAST

DEHYDROGENASE.

Balestrieri,Giovanni

di Chimica

REACTIVE

I Facolta

16, 80138

and Gaetano

Irace.

di Medicina,

Napoli,

Universita

di

Na-

Italia.

4,197s

SUMMARY - The reaction of yeast alcohol dehydrogenase with iodoacetate produces a loss of 90% of the enzymatic activity when 2.2-2.4 equivalents 94 carboxymethyl groups are incorporated. After CNBr cleavage of the C-carboxymethylated enzyme the more radioactive fragment was maleylated and digested with trypsin. A tryptic peptide, containing about 70% of initial radioactivity, was purified and sequenced. The cysteine found to be more reactive with iodoacetate in our experimental conditions is different from that found in previous works. It also appears to differ from the cysteine residues found to be reactive towards other thiol reagents.

Yeast tical

alcohol

or highly

weight

similar

35,000

(1,2).

enzyme binding single

the coenzyme.

sulphydryl

groups Following

Dickinson

(8)

enzymatic

activity.

also

showed

nase and the

(3,4,5)

and Bruni

that

(7)

More

recently, binding

the reaction

of the native

that

Similar buthyl

only

isocyanate is

with

sequence

sites

two thiol

findings

were inactivate

two cysteine of amino

that

acids

a

necessary

the

for

"essential"

the

the number

on co-

contains

group

do not serve

however,

iden-

molecular

each subunit

showed

dehydrogenase

of four

and studies

sulphydryl

and coenzyme

reaction

in the same unique

experiments

and a single

concluded

that

of approximate

site

alcohol

composed

chains

proposition

Auricchio

of yeast

a tetramer

to the

binding

activity.

-SH groups binding

led

is

polypeptide Inactivation

(6)

coenzyme

enzymic

dehydrogenase

function

of

of essential

have been re-examined. enzyme with groups

reported yeast residues, in the

iodoacetate,

are essential

for

by Twu et al.(g) alcohol

dehydroge-

each of which protein

,who

(10).

The

is

Vol. 66, No. 3, 1975

inactivation found jor

BIOCHEMICAL

of yeast

to involve

four

radioactive

with

alcohol

alcohol is

different

study

we report

that

both

by Twu and Wold

from

that

with

sed from

Boehringer,

U.K.).

14

Iodo(2-

The reaction

both

was determined

activity

measurements acid

drochloric

acid,

scintillation

Under

re found

per

with

the

tion

was performed

cold

iodoacetate

sulphydryl of enzyme

and that

for

excess

found

a 16 fold

promptly

assayed

were

mixed

freed

several

at zeEnzyme

(11).

with

Radio-

acetone-

of excess

changes

mo-

Amersham,

and Sund

reagent

of acetone-hy-

The samples

was assayed

2.2-2.4

were

redissolved

in a Tri-Carb

liquid

alcohol

10% active

2.5 hours of in14 C)acetate weof iodo(2-

moles

assuming

dehydrogenase

iodoacetate

of non-radioactive

and to be in a two-fold a number

excess

901

to react

carboxymethy*laiodoacetate;

the excess

of 36 sulphydryl

(1).

dithiothreitol

and complete

with

a molecular

(1,12).

enzyme was added

an excess

assuming

% after

dehydrogenase,

was so much as to react

groups,

buffer,

and radioactivity.

with

alcohol

of radioactive

groups

(purcha-

(Packard).

yeast

using

were

by Wallenfels

radioactivity

with Centre,

Samples

activity

The samples

conditions

- To the

thiol

residue,

dehydrogenase

of the enzyme was 9.6-11.4

these

of 140,000

threitol

(1)

Radiochemical

down and dried.

mole of yeast

CNBr cleavage

a cysteine

was incubated

at 0°C.

precipitates

spectrometer

cubation.

of yeast

in 4 x 10s2M phosphate

made on samples

and the

The activity

out

as described

centrifuged

acid

(The

enzymic

(95:5,v/v).

the obtained

98% formic

for

were

by washing

weight

of 15 mg/ml, C)acetate

activity

in

inactivation

alcohol

dissolved

was carried

times

hydrochloric

(1).

RESULTS

- Yeast

Mannhein),

of iodo(2-l4

ro and later

the

by Harris

AND

C)acetate

at a concentration

excess

reaction

(10).

Reaction

lar

after

by Harris

involves

found

EXPERIMENTAL

pH 7.0,

Two ma-

to the butylcarbamoylated

isolated

iodoacetate

was

(8,lO).

by Twu et al.(g)

to that

with

iodoacetamide

of enzyme

similar

present

dehydrogenase

which

per mole

obtained

RESEARCH COMMUNICATIONS

with

one corresponding

and the other In the

groups were

(14C)iodoacetamide,

peptide

dehydrogenase

thiol

peptides

AND BIOPHYSICAL

over

the

of dithiothe

groups

protein per mole

Vol. 66, No. 3, ? 975

The reaction 8 M urea,

at pH 8.5, The excess

changes genase

of HCl,

BIOCHEMICAL

AND BIOPHYSICAL

was carried

out

under reagents

in the presence for

3 hours

removed

of 0.33

M Tris,

at room temperature.

by dialysis

carboxymethylated

against

yeast

several

alcohol

dehydro-

was freeze-dried. carboxymethylated

ved in 7 ml of 70% formic added.

Following

with

distilled

tion

into

twice

from water

was allowed

trap.

about

24 hours

was diluted

reagents

were

was then

was separated

of radioactive

at room

20-fold

removed

by evapora-

evaporated

fragment

in two fractions,

5 ml of pyridine-acetate

pH 6.5.

sample

for

bromide

to dryness

by lyophilization.

and purification

material

the

The content

mg) was dissol-

250 mg of cyanogen

to proceed

and the excess

ice

enzyme (150

and about

incubation,

water a dry

Isolation

acid

The reaction

temperature.

with

were

pH 3, and the

The freeze-dried

were

nitrogen

RESEARCH COMMUNICATIONS

buffer

by four

consecutive

(10% pyridine,

of the pooled soluble

The radioactivity 90 mg of protein)

was found

- The lyophilized

0.5% acetic

fraction

to be 28-31%

extractions

of the

acid),

(containing initial

radio-

activity. Both the scale

experiment

pH 8.0 paper

for

soluble were

4 hours.

electrophoresis

and insoluble digested

The resulting at pH 6.5.

zed by autoradiography.

sodium de.

fraction

pyrophosphate,

adding

pH 9.0,

about

by additions

60 mg of maleic

x 110 cm),

equilibrated

tion

was carried

out with

of 5 ml fractions).

aliquots

subjected

The radioactive

and allowed

to high

voltage

locali-

were

shown in

fig.1.

in 4 ml of 8 M urea-O.1 to react

as described

a small

at 37°C and

peptides

scheme is

anhydride.

sample

(2.5

with

digestswere

from

with

maleic

M

anhydri-

by Butler

et a1.(13,14)

The pH was kept

at about

by 9.0

of 1 M NaOH.

The maleylated

tored

by incubation

was dissolved

was performed

obtained

trypsin

A qualitative

The insoluble

The reaction

with

fractions

was trasferred with

The absorbance

of the

II,

and the

second

tube

in a liquid

902

G-100

M ammonium bicarbonate.

the same solvent

a LKB 8300 Uvicord from every

0.05

to a Sephadex

at 15 ml/hr fractions radioactivity scintillation

column The elu-

(with

collection

at 280 nm was moniwas followed spectrometer.

on

Vol. 66, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I

+

sol

ins

Fig.1 - Radioautogram of radioactive peptides produced by tryptic digestion of the soluble (sol) and the insoluble (ins) fractions obtained aft75 CNBr cleavage of yeast alcohol dehydrogenase inactivated by iodo (2C)acetate and fully carboxymethylated. Electrophoresis was performed in pyridine-acetic acid buffer at pH 6.5.

The elution

pattern

is

shown

The fractions, freeze-dried. nate applied 0.05

containing

The dry

and digest

with

buffer

residue trypsin

to a column M amnonium

the

for

collection

at 280 nm and radioactivity

were

insoluble

fraction,

containing emerged

The more radioactive described tained

by Butler by high

voltage

G-50

from

at 37°C.

(2.5

paper

The tryptic

x 110 cm),

out with

the column were A single

electrophoresis

903

and

digest

above.

radioactivity

was

with the same

Optical

as described

the 80% of the

pooled

equilibrated

was carried

followed

were

M ammonium bicarbo-

of 5 ml fractions).

fractions

et a1.(13,14).

peak,

in 0.05

4 hours

The elution

(with

peak of radioactivity,

radioactive

was dissolved

of Sephadex

bicarbonate.

at 20 ml/hr

in fig.2.

density One single of the

(Fig.2). pooled

and demaleylated

radioactive at pH 6.5.

peptide

as was ob-

Vol. 66, No. 3, 1975

BIOCHEMICAL

FRACTION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(TUBE ~uMm7)

Fig.2 - Isolation of the main radioactive peptide from iodoacetate inactivated yeast alcohol dehydrogenase. After inactivation the sample was fully carboxymethylated and subjected to CNBr cleavage. The digest was divided in two fractions as described in the text; the insoluble fraction was maleylated and applicg into a Sephadex G-100 column (2.5 x 110 cm) equilibrated with 5 x 10 M ammonium bicarbonate. The elution was carried out with the same solvent at 15 ml/hr with collection of 5 ml fractions (A). The main radioactive fractions were pooled and subjected to tryptic hydrolysis. The tryptic digest was then fractionated by Sephadej G-50 gel filtration. The column (2.5 x 110 cm) was eluted with 5 x 10 M ammonium bicarbonate at 20 ml/hr with collection of 5 ml fractions (B).

Amino

acid

sequence

of the

radioactive

no Acid

Analyzer

of radioactive peptide

after

(6 N HCl,

24 hours,

CMCys(2),

Asp(l),

unhydrolyzed

peptide.

- The amino

in evacuated

The estimated

Pro(l),

Gly(l),

was detected

amino

Ala(2), by using

acid

Val(l), Ehrlich

determined

The N-terminous,

composition

on a Beckman Model

of the sample

(15).

acid

120 C Amiglass

tube

content

was :

Phe(l),

Lys(1).

test

by dansyl

(16)

in the

procedure

was proline. Both

digestion dure

hydrolysis

Ser(l),

of tryptophan

(17),

was determined

105°C)

No residue

peptide

were

(18).

the whole

peptide

subjected

Dansyl-amino

and the

to sequence acid

chromatography

on polyamide

and Wang (19).

The sequences

two peptides analysis

were

identified

sheets

using

were

:

904

by the

obtained

dansyl-Edman

by two-dimensional the

solvents

by its

reported

tryptic proce-

thin

layer

by Woods

Vol. 66, No. 3, 1975

BIOCHEMICAL

14

Pro-Ala-Gly-Ala-Lys-

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

CMCys-CMCys-Ser-Asp-Val-Phe

T1 T2 T The C-terminal like ger

cleavage. peptide

phenylalanine

This

peptide

produced

is

could

probably

represent

formed

the

by a chymotryptic-

N-terminal

part

of a lar-

by CNBr cleavage. DISCUSSION

Using Harris

(l),

approximately we isolated

acid

sequence

yeast

alcohol

of activity

a radioactive

different

from

dehydrogenase was about

(2-14C)acetate that

that with

90% with

was clearly

the

peptide.

found

which

which

by Harris

alcohol

revealed after

having

contained

an amino

of 2.2-2.4

that

of

the

loss

moles

of iodo

we found

we followed

more

and sequenced,

the 80% of radioactivity

about

by

inactivation

dehydrogenase,

The peptide,

employed

Even if

an incorporation

more radioactive,

fraction

peptide

conditions

iodo(2-14C)acetate.

per mole of yeast

one radioactive

insoluble

the same experimental

the 70% of the

of the

initial

radioacti-

vity. The peptide,

that

we sequenced,

amino-terminal

region

of the

tly

in the

primary

identified

peptide

with It

14

the is

C-labeling.

acetate

et al.(Zl)

dehydrpgenase cyanate) mined

with

reagents

different

by the pH at which iodoacetate but

at lower

and 7.5

the reaction is

is

incorporated

pH values

the

alcohol

labeling 905

has recen-

dehydrogenase

localization

of with

iodoacetamide,

out;

preferably increases

for

Recently

of yeast

in an amount

iodo-

except

in Harris'work.

carried

a

result).

the inactivation

residues

in the

worker

conditions,

(iodoacetate,

cysteine

this

dehydrogenase

experimental

that

by Jornvall

unpublished

alcohol

in our experiments

thiol

however

the different

of yeast

have demonstrated

involves

radioactive pH 7.5,

was 7.0

explain

in similar

found

of yeast

(H.Jornvall,

not easy to fully

out

(20);

structure

The inactivation

the pH, that Belke

molecule

same sequence

was carried

was not

alcohol

butyl strongly

in particular

in the

isodeterthe

Harris'peptide

in the

butyl

at isocya-

Vol. 66, No. 3, 1975

nate-reacting ferent

peptide

segments

cleavages

of the

Twu et al.(g)

cule

exposes

difications

and in other

peptides,

Harris'peptide

results

and Belke

contains

varying

AND BIOPHYSICAL

that

produced

RESEARCH COMMUNICATIONS

were

as dif-

considered

by heterogeneous

peptic

(21). The present

genase

BIOCHEMICAL

et a1.(21)

more than to various

thiol

to conformational

groups changes,

than

thiol

reagents

group

impair

points

Dickinson alcohol

(8), dehydro-

and the enzyme molecysteine

and of the

in different

(l),

the yeast

different

conditions

which

of Harris

indicate

one reactive

of the experimental of thiol

and the works

residues

employed

of the

more or less

reagents.

protein

give

the enzymatic

with

the

Morise acti-

vity.

REFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21)

Harris,J.I.(1964) Nature 203, 30-34. Butler,P.J.G.,Jornvall,H. & Harris,J.I.(1969) Febs Letter 2, 239-241. Rabin,B.R. & Whitehead,E.P.(1962) Nature 196, 658-660. Whitehead,E.P. & Rabin,B.R.(1964) Bi0chem.J. 90, 532-538. Rabin,B.R.,Cruz,J.R.,Watts,D.C. & Whitehead,E.P.(1964) Bi0chem.J. 90, 539-546. Hayes,J.E. & Velick,S.F.(1954) J.Biol.Chem. 207, 225-244. Auricchio,F. & Bruni,C.B.(1969) Biochim.Biophys.Acta 185, 461-463. Dickinson,M.(1974) Eur.J.Biochem. 41, 31-36. Twu,J.,Chin,C.C.Q. & Wold,F.(1973) Biochemistry 12, 2856-2862. Twu,J. & Wold,F.(1973) Biochemistry 12, 381-386. Wallenfels,K. & Sund,H.(1957) Pi0chem.Z. 329, 17-30. Buhner,M. & Sund,H.(1969) Eur.J.Biochem. 11, 73-79. Butler,P.J.G.,Harris,J.I.,Hartley,B.S. & Leberman,R.(1967) Bi0chem.J. 103, 78P. Butler,P.J.G.,Harris,J.I.,Hartley,B.S. & Leberman,R.(7969) Bi0chem.J. 112, 679-689. Spackman,D.H.,Stein,W.H. & Moore,S.(1958) Analyt.Chem.30, 1190-1206. Smith,I.(1953) Nature 171,43-44. Gray,W.R.(1967) "Methods in Enzymology" 11, pp.139-1Sl - Academic Press, New York. Gray,W.R. & Hartley,B.S.(1963) Bi0chem.J. 89, 379 and 59P. Woods,K.R. & Wang,K.T.(1967) Biochim.Biophys.Acta 133, 369-370. Jornvall,H.(1973) Proc.Nat.Acad.Sci.U.S.A. 70, 2295-2298. Belke,C.J.,Chin,C.C.Q. & Wold,F.(1974) Biochemistry 13,3418-3420.

906