Esterase activity of liver alcohol dehydrogenase

Vol. 86, No. 3, 1979 February

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

Pages 808-814

14, 1979

ESTER&E

ACTIVITY

OF LIVER ALCOHOL DEHYDROGENASE C.S.

Department

of Chemistry

Carleton Received

December

Tsai

and Institute

University,

Ottawa,

of Biochemistry Ontario,

Canada

22,1978

SUMMARY Liver alcohol dehydrogenase is found to possess, in addition to its dehydrogenase and dismutase activities, the ability to hydrolyze octanoate esters at a rate approximately l/500 - l/1000 of that of The esterase and dehydrogenase activities the dehydrogenase reaction. exhibit an identical isozyme pattern indicating that the same protein catalyzes both reactions. Inhibition studies suggest that the esterase activity presumably shares the catalytic domain with the dehydrogenase activity. INTRODUCTION Alcohol

dehydrogenase

from horse.liver

(LADH*)

The enzyme catalyzes primary

as well

as secondary to mediate

acids

However,

reaction

is questionable

to acids

is

exhibit

at pH 7.4, activity

of LADH.

LADH increases

with

a maximum at octanal

IAbbreviations nitrophenyl benzoate.

of aliphatic,

alcohols

(1).

the dismutation

(7)

failed

In view of the an increase with

in

a higher

oxidation

that

optimum

of aldehydes

dehydrogenases

p-nitrophenyl

carbon

the enzyme has

of the dismutase

to demonstrate

fact

and aromatic

significance

of aldehyde

and Neal

alicyclic

specificity.

to the corresponding

by a group

Using

substrate

of aldehydes

The physiological

(5,6).

EC 1.1.1.1)

In addition,

(3).

activity

Hinson

by a broad

the physiological

catalyzed

esterase

oxidoreductase,

is characterized

the oxidation

been reported (2,3).

(alcohol:NAD'

the chain

(4)

acetate

which

as a substrate

the esterolytic

dismutase

activity

of substrates

pH, we tested

the

of

to reach ability

used are: LADH, liver alcohol dehydrogenase; NPA, pacetate; NPO, p-nitrophenyl octanoate; PCMB, p-chloromercuri-

0006-291X/79/030808-07$01.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

808

also

Vol.

86,

No.

BIOCHEMICAL

3, 1979

of LADH to hydrolyze demonstrate

octanoate

the esterolytic

AND

esters

BIOPHYSICAL

RESEARCH

at pH 8.2 and were

activity

COMMUNICATIONS

able

to

of LADH.

MATERIALS AND METHODS Alcohol nitrophenyl were

dehydrogenase, octanoate

obtained

(NPO),

from Sigma

of K & K Laboratories. Eastman

Organic

sodium

prior

(CM-52,

weight

were

were

purchased

separated

from

dioxane were

against

obtained

distilled

water

by CM-cellulose

as described

(8).

1% cIcrn

from A280 taking

p-

was a product

o-phenanthroline,

chromatography

The enzyme

= 4.2 and a subunit

(9).

activity

activity

the rate

of p-nitrophenol

mixture

(2% dioxane)

containing M borate

Elmer

spectrophotometer

To correct

identical

reaction

dissolved

in dioxane

the

buffer,

rate

was carried

by

mM NPO and 5.0 _+ 2.5

equipped

a variable

hydrolysis

in the absence

solubility

UM LADH

a Beckman DB or Perkinwith

of spontaneous

due to its

(10).

at 400 nm of a reaction

pH 8.2 with

out

as described

spectrophotometrically

release 0.10

in 1.0 ml of 0.10 124)

of LADH was assayed

of LADH was assayed

following

recorder.

octanoate

and cadmium acetate

Isozymes

The dehydrogenase

(model

Ethyl

(PNA),

(pCMB) and iodoacetate

LADH was dialyzed

was estimated

The esterase

acetate

and pyrazole

thiocyanide

to use.

of 42,000

Co.

p-Nitrophenol,

Co.

Whatman)column

concentration

Chemical i-Butyramide

Scientific

overnight

p-nitrophenyl

p-chloromercuribenzoate

Chemicals.

(spectrograde), from Fisher

NAD',

output

of NPO, an

of LADH.

and to minimize

NPO was the spontaneous

hydrolysis. The titrimetric out by following (6.0

ml)

containing

assay the rate 0.10

of the esterase

of acid M

KCl,

liberation 0.20

LADH by means of a pH-stat

(Radiometer

All

at 30?2'C.

assays

were

maintained

mM ethyl

activity

of a reaction octanoate

TTTla/SBR2c/SBUl)

809

was carried mixture and 5.0

$I

at pH 8.2.

Vol. 86, No. 3, 1979

TABLE

I.

BIOCHEMICAL

Spontaneous

and

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

LAD-l-catalyzed

Hydrolysis

of

v(uncorrected Acid

Reaction

Liberated

Ethyl

Octanoate

rates)

(PM min-I)

NADH formed (uM min -5 0

Et-octanoate

alone

0.833

Et-octanoate

+ NAD+

1.50

Et-octanoate

+ LADti

3.06

0

Et-octanoate

+ NAD+ + LADH

3.35

4.18

NAD+ + LADH

0

0

0.563

Rates of hydrolysis of ethyl octanoate (200 PM) in 0.10 M KC1 were measured at pH 8.2 in the presence or absence of LADH (5.0 uM) and/or NAD+ (1.0 mM).Octanoic acid liberation was measured titrimetrically by the base (1.0 mE1 NaOH) consumption and ethanol formation was followed spectrophotometrically at 340 nm according to the reaction sequences: Ethyl

octanoate

-Ethanol

+ Octanoic

NAD+

acid

NADH + H+

-L Acetaldehyde

RESULTS AND DISCUSSION An aqueous hydrolysis

and

(Table by

-via of acid

1).

the

rate

octanoic

shows

accompanied

by

p-Nitrophenyl

found

assay

to

hydrolyzed since

the

increases

for

The

and specific

of the of

liberation of

rate activity

Fig. of

LADH

LADH

1 mole

that

to

monitored indirectly

the

this

810

NAD'

by

ethanol

direct

produced.

spectro-

have been

common substrate

(7).

At

catalyzed

to reach readily

NPO at

NPA was

surprising dismutation

the optimum hydrolyzed

to

was

pH 8.2,

hardly

is

proportional

hydrolyze

octanoic

of dehydrogenases

LADH

chain

hydrolysis

of

of

This

NPO was

was

were

1 mole

and

pH 7.4

of NPO. in

hydrolysis of

at

in carbon

1 shows

of

However

rate

LADH

production

a convenient

aldehydes

an increase

octanoate

ethanol

A number

by

by

of the LADH catalyzed

provide

hydrolyzed

(3). the

the

spontaneous

facilitated

and

NPA (5,11,12).

reactivity

with

of ethyl

esterases.

at l/3-1/4

at octanal LADH

be

products

reduction

esters

shown to hydrolyze not

was

that the

underwent

hydrolysis

The stoichiometry

octanoate

photometric

octanoate

acidliberation

NADH formation.

is

of

The hydrolysis

following

ethyl

of ethyl

solution

LADH pH 8.2

by concentrations. is

l/500-l/1000

Vol. 86, No. 3, 1979

BIOCHEMICAL

Time

I 0

1 0

Fig.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(min)

I .4

I 1 .a 1.2 NAD+ (mM) I 1 1 .04 .00 .I2 p-Nitrophenyloctanoate

1.

Hydrolysis

L I J 1.6 2.0 2.4 (a) 1 I I .I6 .20 .24 (mM1 (0)

of NPO by LADH

Effect of LADH (O), NAD+ (A) and NPO (a) on the rates of p-nitrophenol release in O.lOM borate buffer, pH 8.2 were measured spectrophotometrically at 400 nm. Experimental conditions were: (O), 100 PM NPO with varied LADH concentrations (A), 5.0 PM LADH + 100 PM NPO with varied NAD+ concentrations (a), 5.0 PM LADH with varied NPO concentrations All rates were corrected for spontaneous hydrolysis. Inset shows time courses for p-nitrophenol release at pH 8.2 for spontaneous hydrolysis (I, NPO = 100 PM), LADH (2.5 IJI) catalyzed hydrolyses of NPO (100 FM) in the absence (O).and presence (A) of NADt (O.lOmM).

that

of the

hydrolyses

dehydrogenase of octanoate

the co-enzyme

esters

were

was not essential

for

To examine activity

the molecular

slightly

chromatography

which

2 shows the intimate

and dehydrogenase

identity

between

activities

presumably

was subjected

separates

four

correspondence

activity derive

for

all from

enhanced

the esterolytic

and LADH, the dehydrogenase

column Fig.

Rates of spontaneous

activity.

by NAD', activity

between

IAiM.

to a CM-cellulose isozymes.

the esterase

suggesting

the same protein

of

however

the esterolytic

ethanol-active

isoyzmes

811

and LADH catalyzed

that

molecule.

activity the two

Vol. 86, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6-

S-

3 0 z a 2-

I-

O,

0

IO

20

40

30

50

FRACTIONS Fig.

2.

CM-Cellulose

60

70

0

80

(2.6 ml / FRACTION)

Column Chromatography

of LADH

Ten mg of LADH was dialyzed against 2.0 mM phosphate buffer, pH 7.0 overnight and delivered onto a CM-52 column (Sephadex K9/30). The enzyme was eluted with a linear gradient of an increased phosphate buffer (pH 7.0) concentrations from 2.0 mM to 20 mM. Buffer concentrations (---) of eluates was estimated from the conductivity and protein (-) was determined at 230 nm. The dehydrogenase (0) and esterase (a) activities were assayed spectrophotometrically at 340 nm and 400 nm (NPO) respectively as described in the text. To further for were

substantiate

the esterase tested

activity,effects

(Table

to the active-site

Znsf

(13)

by a decreased

esterolytic

and i-butyramide

which (15,16),

Treatment

in an inactivation of sulfhydryl It and alcohol

is

competed likewise,

of LADH with

with

noted

that

dehydrogenase

for

both

activity

ethanol

inhibited iodoacetate activity

which

of LADH.

was also

demonstrated

LADH (14).

and acetaldehyde the

LADH catalyzed

(17)

chelate

or pCMB (14)

implicating

for

Pyrazole LADH

hydrolysis results

the requirement

activities.

the esterase differs

of LADH

activity

of Cd“+ - replaced

of the esterase group(s)

the esterase

the esterase activity

of inhibitors

and thiocyanide

inhibited

for

the enzyme responsible

of a number

o-Phenanthroline

of Zntf

of NPO.

LADH is

2).

The requirement

respectively

that

activity in several

812

of aldehyde aspects.

dehydrogenase NAD' which

greatly

Vol. 86, No. 3, 1979

BIOCHEMICAL

II.

Table

Effect

of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

LADH Inhibitors

specific Inhibitor None

on Esterase

Activity

activity

(PM min -1 mg -1 ) (control)

% Inhibition

2.51

l.OpM

Pyrazole

0.752

500uM

i-Butyramide

1.75

50pM o-Phenanthroline

1.25

50~M

1.50

thiocyanide

0.05mM Cadmium acetate (2m.) 2.5mM Iodoacetate (10 min.)

0.752

0.50mM

1.25

pCMB

(15

69.9 30.3 50.2 40.2 60.2 69.9 50.2

1.00

min)

Alcohol dehydrogenase (5.0 PM) catalyzed hydrolysis of NPO (100 MM) in 0.10 M borate buffer, pH 8.2 were studied in the presence of inhibitors at the concentrations given. To study effects of cadmium ion (14), iodoacetate (15) and pCMB (14), LADH was incubated with these reagents at the concentration indicated for the specific time periods prior to assay. All rates were corrected for spontaneous hydrolysis.

promotes only

the hydrolytic

a slight

enhancement

dehydrogenase. hydrolysis genase

reactions (18).

as esterase

octanoate

whereas

same

has

Zn++ is essential

domain

at pH 8.2, both

for

the

ester

both

to its

aldehyde

aldehyde for

activity

dehydrofor

of

as well

activity

activities. toward

oxidation-reduction studies

presumably

activity. furnished

at the

LADH.

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