Vol. 81, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

March 30,1978

Pages 422-426

COENZYMEBINDING CAPACITY OF YEAST ALCOHOLDEHYDROGENASE

V. Leskovac and D. PeriEin Department of Chemistry,

P.O.Box

187, University

of Novi Sad

21000 Novi sad, Yugoslavia Received

January

24,1978

Summary Commercial lyophilized preparations of yeast alcohol dehydrogenase from Boehringer G.m.b.H. (Mannheim, Germany) bind 2 mols of reduced coenzyme/144000 g of enzyme ( 1 ). After the purification by a DEAE-Se phadex column chromatography, the coenzyme binding capacity is raised to 4 mols of NADH/mol of enzyme. Commercial preparations and ionexohange-purified preparations are homogeneous on the ionexchange column chromatography and the disc gel electrophoresis, after reduction with thioglycolic acid. Ionexchange chromatography does not increase the -SH titer,, zinc content and the specific activity of enzyme. It is sugges ted that ionexchange chromatography raises the NADH-binding capacity by removing some impurities present in commercial enzyme preparations.

Introduction Yeast alcohol

dehydrogenase is a tetrameric

four seemingly identical gations

of Dickinson

zyme binding Dickinson (

(

polypeptide

3 ) indicated

sites/tetramer;

was questioned

recently,

enzyme, composed of

chains ( 2 ). Extensive that

the enzyme has only 2 coen-

the binding

by Temler & Ktigi (

capacity

investigated

ger/Mannheim,

commercial lyophilized

and found that

NADH/aol of enzyme ( 1,6,7 sence of two additional

they invariably ). In this

binding

found by

4 ) and by XarloviO

5 ), who have found 4 coenzyme binding sitesltetramer.

matically

investi-

preparations bind approx.

We have systeof Boehrin2 mols of

communication we report

sites/tetramer

et al.

the pre-

of the commercial pre -

paration,

which are unmasked by the ionexchenge column chromatography.

Materials

and.Methods

Lyophilized yeast alcohol dehydrogenase (EC 1.1.1.1) was purcha sed from Boehringer G.m.b.R. (Mannheim, Germany). !:nzyme activity assay, 000~291X/78/0812-0422$01,00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

422

8lOCHEMlCAL AND 8lOPHYSlCAL RESEARCH COMMUNICATIONS

Vol. 81, No. 2, 1978

determination of protein concentration and spectrophotometric measure menta were performed as previously described ( 1,6,7 ); determination of coenzpe binding oapacity by the fluoreecent titration and the purifi cation of commercial preparations by the DEAE-Sephadex column chromatography was performed as described elswhere ( 1 ). Disc gel electropho resis was performed according to Brewer & Ashvorth ( 8 ), and the reactivity of enzyme -SH groups was followed by the method of Bllman ( 9 ), in a manner described earlier ( 1 ).

Results

and Discussion Commercial

dehydrogenase

preparations are homogeneous

phy ( 1 ).

Both commercial

show three

distinct

ing with

on the DEAR-Sephadex

bands on disc

After

reduction

specific

gel electrophoresis

of enzyme, not

obtained

with

exceeding

-SK groups

the state

, 8 zinc

used

titer,

and the specific

enzyme binding 2) are

capacity changed

Commercial of NADH/mol bind

(Fig.

on the Figure

preparations

NASH were

l),

-

both

show a single and the

indicates

the ho-

of oxidation,

since

the reversal

have had 20 - 24 free

and a specific did not

of

change the -SH

of enzyme.

Only the co-

of -SH groups

invariably

1

approx.

ionexchange-purified 1).

. Concentrations at each titration

423

2 mols

preparations

Coenzyme binding

by an appropriate

that

activity

chromatography.

of enzyme bind

VS* l/batota assuming

work

and the reactivity

of enzyme (Fig.

1 was determined

determined

acid

activity

of enzyme ( 1 ) , whereas

Of l/b~Hbom~

stain

acid,

states

chromatography

by the column

4 mols of NADH/mol

This

in variable

atoms/molecule

Ionexchange

content

preparations

amide-schwarz

( 8 ).

in this

380 U/mg at pH 9.0. zinc

chromatogra-

electrophoresis,

with

of sulfenic

alcohol

acid.

preparations

-SH groups/molecule

column

preparations

mixture

by thioglycolic

Commercial

yeast

by thioglycolic

, staining

dehydrogenase-staining

mogeneity

(Fig.

gel

( 1 ) and ionexchange-purified

band on disc

probably

Boehringer

( 1 ) and ionexchange-purified

protein

amido-rchwarz.

commercial

is

of lyophilized

linear

capacity

extrapolation

of enzyme-bound point,

I

-

Vol. 81, No. 2, 1978

8lOCHEMKAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

012

046

W

I 1.0

08

l/L total

FLg. 1. Coenzyme binding capacity of purified yeast alcohol tlehy droizenase, determined by the fluorescent titration. In the forward ti tration (&cesented on the figure), the enzyme (1.25 )IM, mol. wt. 144000) was titrated with NADH (0 - 9.1 JGT) in 0.1 M sodium phosphate buffer, pH 7.3 (with 0.5 3. EDl?A and 0.5 M acetamide final volume 3 ml. In the reverse titration (net shown), NADH (0.91 @-? was titrated with the enzyme (0 - 3.66@l) in the same buffer. Different symbols represent different enzyme preparations. Coenzyme binding capacity (4.0 mols of NADH bound/m01 of enzyme) was obtained by dividing the concentration of bound coenzyme at saturating NADH-concentrations ( fLboundl = 5 pM) with the concentration of enzyme (1.23 p).

d#UDHfree]

+p[NADHbound],

ces of

and bound coenzyme , and I,

given

trating

free titration

point;

where d.

and /$

are specific

the total

fluoreecen-

fluorescence

/3 was determined in a reverse

in a

titration,

the coenzyme with the enzyme. Thus the determination

by tiof

, usually problematical in fluorescent titrations, was free INADEboun(5 of assumptions based on values easily determined with precision. Ionexchange chromatography

changes the reactivity

groups towards 5,5'-dithiobis-(2-nitrobenzoic 12 - 20 -SH groups/molecule bis-(2-nitrobenzoate)

at

a

of enzyme react uniform

reaction

acid) with rate,

of enzyme (Fig.

2). Initially,

excess of 5,5'-dithiowith k2 = 25 M-lmin-'

in the commercial and k2 = 66 M-'lain -1 in the ionexchange-purified

424

thiol

pre-

8iOCHEMlCAl AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 81, No. 2, 1978

-6.5 -60 0

2

L

0

6 Time

IO

-

12

I 4 Time [mid

2

imid

6

Fig. 2. Reactivity of enzyme -SH groups towards 5,5’-dithiobis(2-nitrobenzoate). A: Purified enzyme preparation (3.9 @, mol. wt. 144000) was mixed with 3.25 mM) in 0.1 M Tris-BCl buffer, 5,5’-dithiobis-(2-nitrobenzoate pH 8.6, in the presence (160 @I H l ) and in the absence of NADH ( o ); was displayed on a recorder, and chart records were evaluated ac:8ha ing to the method of Guggenheim ( 10 ). B: Commercial preparation (5.25 @I) was mixed with 5,5’-dithiobis-(2-nitrobenzoate) (3.25 n@?)in the same buffer, in the presence (160~) or absence of NADH, and A evaluated as above. 412

paration, binary

respectively,

at pH 8.6.

In the commercial preparation,

complex enzyme-NADH, reactivity

of approx.

towards 5,5’-dithiobis-(2-nitrobenzoate) j-fold

(k2 = 72 M-lmin-l)

2-fold

sensitization

with respect

enzyme to this

of all

in comparison with

-SH groups is

the non-liganded

2).

We assume that

the increase

in coenzyme binding

from the removal of some impurity

by the ionexchange

This impurity

and enhanced

by coenzyme is abolished)

complex enzyme-NADH, the reactivity

lower ~(k2 = 33 I&zin-‘)

enzyme (Fig.

is sensitized

enzyme is desensitized

phenomenon, and the selective in the binary

2 -SH groups/molecule

, in comparison with the non-liganded

( 1 ). Ionexchange-purified

in the

should represent

capacity

arises

chromatography.

less then lO$ of the commercial material,

425

Vol.

61,

No.

BIOCHEMICAL

2, 1978

since larger

impurities

chromatography nucleotide

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

should have been detected

or gel electrophoresis;

impurities

es to stabilize

or a protein

the likely stabilizer,

commercial preparations

Our finding

that

the binding

either

by the column

candidates

are some

added in small quantiti-

( 11 ).

capacity

of a nearly

homogeneous pre-

paration

was raised from 2 to 4 mols of IUDH/mol of enzyme, without

increase

in specific

dicates

that

activity,

raises

an intriguing

the enzyme may bind maximally

substantiated (

j

ascribed

usually

) and tightly

active

sites/tetramer.

to 'half-the-sites-active'

by half-the-site

reactivity

rent half-the-sites

data are clearly

reactivity

enzymes, is

of essential

bound zinc atoms ( 7 ) , reported

However, more experimental

This in -

4 molecules of coenzyme, pos-

sessing at the same time only 2 catalifically This property,

question.

the

thiol

for

groups

this

enzyme.

needed to clarify

of yeast alcohol

the appa-

dehydrogenase.

References 1. Leskovac, V., 2, 000-000. 2. Jbrnvall,

II.

3. Dickinson, 4.

PeriEin, (1977)

F.M.

D. and Trivid,

Europ.

S. (1978)

J. Biochem. j'2,

J.H.R.

(1974)

D., Amiguet, P., Bonner, F.J. Europ. J. Biochem. 66, 277-284. V. and Pavkov-PeriEin,

Leskovac, V.,

Trivid,

and Ashworth,

9. Ellman,

(1959)

10. Guggenheim, E.A.

Arch.

R.D. (1963)

M. (1976)

426

687.

P.L.

(1976)

82, 70-77.

2433454.

581-590.

Biochem. J. a,

J. Chem. Education

Msg. 2, 538-540.

Offenlegungsschrift

2,

Biochem. J. l&~,

Biochem. Biophys.

(1926) Phil.

11. Deutsches Patentamt,

and Luisi,

D. (1975)

S. and Latkovska,

8. Brewer, J.M. G.L.

425-442.

Experientia

5. Karlovid,

7.

J. Biochem.

Europ. J. Biochem. 41, 31-36.

(1974)

Temler, R.S. and K1gi,

6. Leskovac,

Int.

&,

41-45.

155-161.