Unusual kinetic properties of a DPN-linked lactate dehydrogenase from Butyribacteriumrettgeri

Vol. 22, No. 6, 1966

BIOCHEMICAL

UNUSUAL KINETIC

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PROPERTIES OF A DPN-LINKED

DEHYDROGENASE FROM BUTYRIBACTERIUM

Charles

L. Wittenbe

LACTATE

RETTGERI

‘r

U. S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, National Institute of Dental Research, and the American Dental Association, Bethesda, Maryland

Received

February

21, 1966

A DPN-linked from

extracts

lactate

of Butvribacterium

stereospecificity kinetic 2. reftgeri

other

LDH is where

sources.

reduction moidal

this

with

reaction

obtained

with

will

glucose

approximately

Company.

enzyme,

reduction

cofactors

substrate

some unusual report

of DPN with readily

be shown that

the

the

lactate

with

rate

that

LDH's

under from

of pyruvate

LDH, increases

in a sig-

of pyruvate. employed

in this

rabbit

muscle

Crystalline

in a semisynthetic

as the energy previously

the DPN-LDH from crude

cellulose

the

concentrations - All

the

be shown in this

by the 2. rettgeri

of 2. rettzeri

as described

accomplished

will

this

purified

study

were

purchased

LDH was a Boehringer

from Calbiochem.

Cultivation 1950)

it

and Methods

for

studying

can be demonstrated

increasing

the Sigma Chemical

It

to catalyze

by DPNR, as catalyzed fashion

product

were

unable

While

requirement

observed.

In addition,

Materials from

were

(LDH) has been partially

rettgeri.

and coenzyme

properties

conditions

dehydrogenase

extracts

a loo-fold

and Flavin,

and its

separation

of ammonium sulfate The details

purified

(Kline

and the preparation

(Wittenberger

by a combination chromatography.

source

medium

of this

enzyme,will

729

and Barker,

of cell-free

1962).

extracts

Isolation

from DPNH oxidase fractionations

of was

and DEAE-

purification,

which

be published

elsewhere.

yields

Vol. 22, No. 6, 1966

BIOCHEMICAL

A D(-)-specific Kaplan, were

1960).

through

in cold

an Aminco

centrifuged

40-80x

X g for

solid

preparations Lactate

dependent Haaf, pyridine

LDH which

All

the

cuvette

a Sargent

specific

for

of the L(+)-isomer pyruvate

as

I).

The enzyme has a specific completely

inactive

The enzyme also oxidation

with

observed

Although

with

a-ketobutyrate, when pyruvate

the 2.

rettgeri

For

pH 8.0, g.

87.3 rettgeri

volume

of

by the addition DB spectrophoto-

LDH from 3.

and its

by the presence

requirement

for

for either

activity

served

with

the

activity is also

DPNH as the coenzyme.

pyruvate

of pyruvate

reaction

D(-)amount of the shown. TPNH is

to lactate.

as the substrate.

D-ketoglutarate

but the

rettgeri

of an equivalent

purposes,

in the conversion

specificity

of DPNH occurred

was oxidized of that

exhibits

1956).

initiated

and

3-acetyl-

of purified

and DPNH as the reductant

as a reductant

of the

The final

DPN-linked

For comparative

substrate

1960).

the pyruvate-

100 r@ Tris-HCl,

were

enzyme.

and Kaplan,

and Ciotti,

per min.

at

(Wittenberger

reduction

of lactate

affected

(Table

of the

by following

in a Beckman Model

- The purified

significantly

precipitated

SRL recorder.

the D(-)-stereoisomer

not

enzyme with

followed Model

and Discussion

which

APDPN, and an amount

was one ml and reactions were

was then

described

contained

was

fluid

LDH (Dennis

lactate-dependent

Cells

extract

as the source

either

and

and passed

The resulting

was assayed

2 +

reactions with

Results

is

activity

at 365 IQ.I of .OZO-.lOO

mixture

equipped

lactate

an L(+)-specific

each reaction

gave ab0.D.

of enzyme. meter

contained

(Dennis

T. Shiota.

pH 6.5,

The fraction

was employed

or Na-L(+)-lactate,

each reaction

two times.

of DPN (APDPN) at 365 rn~ (Kaplan

assay,

a&l Na-D(-)-

buffer,

of DPNH at 340 mu as previously

analogue

by Dr.

20 min and the supernatant

or by measuring

latter

phosphate

cell

plantarum

supplied

saturation

dehydrogenase

1964),

kindly

ammonium sulfate.

also

oxidation

were

pressure

ammonium sulfate

These

is

French

with

cells

from Lactobacillus

10 a@ potassium

at 30,000

fractionated

the

LDH was obtained &. plantarum

suspended

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

No

or oxaloacetate. rate

was only

about

DPNH l/10

as substrate.

LDH catalyzed

730

the oxidation

of lactate

with

Vol. 22, No. 6, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Substrate

I

Stereospecificity

of 2.

Substrate

rettgeri

LDH

Specific+ activity

Cofactor

D(-)-lactate

APDPN

354

L(+)-lactate

APDPN

none

APDPN

343

D(-)-lactate

+ L(+)-lactate

Pyruvate

DPNH

Assay conditions when L(+)-lactate enzyme was used. * wmoles

APDPN as the acceptor easily

with

the L(-+)-specific

tion

readily

It

only

was considered

that

DPN-binding

capacity

APDPN by lactate in Fig. reciprocal

the B. rettgeri

to the &. plantarum activity decreased

for

DPN.

the effect

are

conditions

could

731

no reduca 4-fold muscle

be demonstrated.

ability

of the 2. rettgeri might

reflect

As an indirect

test

of DPN on the

reduction

expressed

may be seen that

lactate,

or rabbit

reaction

experiments

in the Lineweaver-Burk both

where

enzyme from

LDH. Even with

A summary of some of these

the results It

under

of DPN with

of the physiological

was studied.

not be demonstrated

and a D(-)-specific

the markedly

of the enzyme,

plots.

could

shows that

the reduction

of the enzyme

1, where

II

of DPN-reducing

the reverse

low affinity

reaction

muscle

with

and Methods except case twice as much

per min per mg protein.

Table

LDH, compared level

LDH to catalyze

double

this

rabbit

be observed

a trace

relatively

I),

catalyzed

of 2. rettgeri

enzymes,

given

(Table

LDH from

of DPN could

excess

or DPNH oxidized

DPN as the acceptor.

&. plantarum

2,712

were as described in Materials served as substrate, in which

APDPN reduced

detected

DPN and DPNH are

a

of the of is (1934) effective

Vol. 22, No. 6, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table DPN Reduction

II

by Lactate

Dehydrogenases

Source

Sources

Units LDH* assayed

Substrate muscle

by Lactate

from Various

of

enzyme

Rabbit

as Catalyzed

wmoles DPN reducedfmin

L(+)-lactate

50

21.9

_. L plantarum

D(-)-lactate

50

5.7

g.

rettgeri

D(-)-lactate

50

0

2. rettgeri

D(-)-lactate

200

1.1

Assay conditions were as described in Materials and Methods for the APDPN assay except that 2 s&i DPN was substituted for APDPN and reactions were followed at 340 nl.r.

h nits

were determined by assay of the enzymes with DPNH and pyruvate as described in Materials and Methods. One unit is the amount of enzyme which oxidizes one lqLmole of DPNH per minute. The &. plantarum preparation employed here contained both D(-)and L(+)-specific LDH's. In order to estimate what percent of DPNH oxidation by pyruvate was due specifically to the D(-)-enzyme, the preparation was assayed in the presence of an amount of oxamate which inhibited the L(+)-enzyme activity by over 90%, but which was without effect on the activity of the D(-)-enzyme (Dennis and Kaplan, 1960).

inhibitors

of APDPN reduction

competitive neither

with TPN nor

centrations

respect

to APDPN.

TPNH act

as inhibitors

equivalent

The kinetic Table

III.

preparations obtained. values

to those

constants for

and values

ranging

DPN for

values, LDH's

It

from

DPN has varied from

other

inhibition,

has been

shown here

however, from

the

found

in both

cases,

in other

studies

of APDPN reduction

calculated

The Ki value

These for

and that

fall

4.32 well sources.

732

for

is that

when used at con-

DPN and DPNH.

the data

in Fig.

somewhat

with

x 10 -4 _Mto within

1 are given different

7.84

the range

For example,

x 10

-4

enzyme _M have been

of reported the s

in

for

s DPN

Vol. 22, No. 6, 1966

BIOCHEMICAL

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

I

I

I

4

8

12 1

I6

20

24

iiiiixm

Fig.

Effect

1.

were

as described

varied

between

was included

the in the

x 'o-2

of DPN or DPNH on APDPN reduction

in Materials limits reaction

and Methods

shown.

54APDPN 1.85

of)

x 10 -3

except

Where indicated,

by lactate.

APDPN concentrations

Constants

III for

2. rettaeri

Ki DPN

7.84

733

CM)

x 1o-4

Assays were

1 x 10 -4 _M DPN or DPNH

mixture.

Table Kinetic

I

28

LDH

.Ki DPNH

4.55

(Ml

x 10 -4

Vol. 22, No. 6, 1966

BIOCHEMICAL

for

Bacillus

for

rat

liver

LDH is

the 5's

for

DPN for

-4 9 x 10 _M at pH 7.2 (Yoshida, 1965); the value -4 J+! at pH 8.6 (Anderson, et al., 1964); and 1.6 x 10

subtilis

LDH is

various

heart

lo-4

_M to 1.6 x 10 -4 g at pH 8.7

Fig.

1 show that

the

DPN is

therefore,

unlikely,

reduction

of lactate

affinity

of the

lactate

higher

was found

than

the s

D(-)-specific however,

enzyme for

LDH from&. whether

this

Employing _M at pH 8.0.

(with

modestly

of this

x

from

LDH and it

appears

enzyme to catalyze

lowered

affinity

in demonstrating

the 2. rettgeri

lactate.

plantarum

0.88

of

coenzyme.

the difficulty with

lactate

ability

from The data

1964).

due to a markedly

nucleotide

to be 0.11

for

reduced

is

that

et al,,

range

by the 3. rettgeri

bound

lactate

to pyruvate

LDH isozymes

(Nisselbaum,

the greatly

considered

version

for

that

the pyridine

It was also

and brain

effectively

of DPN with

the enzyme for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

LDH might

This

value

and Kaplan,

decreased

affinity

0.0

I 1.2

be due to a low

APDPN as the acceptor,

APDPN as acceptor) (Dennis

the con-

is

about

reported 1960).

of the 2.

the lCk, 3-4 times

for It

rettoeri

the is

uncertain, LDH for

2.02 w Lg 1.6-

0.4 pMOLES

Fig.

2.

Assay conditions concentrations

Effect

of

were were

pyruvate

PYRUVATE

concentration

as described

varied

in Materials

as shown.

734

1 1.6

I 2.0

/ml

on the rate and Methods,

of DPNH oxidation. except

pyruvate

BIOCHEMICAL

Vol. 22, No. 6, 1966

lactate

provides

this

enzyme to catalyze

cofactor.

the

than

has not

demonstrating

Escherichia

coli,

that

concentrations, tration

deviation

This

of kinetic

type

enzymes,,

which

effector

ligands.

has a greatly

reduced

from Michaelis-Menten response

Whether

the conversion

muscle

that

activator

the

(Wolin,

1964).

kinetic

properties

inability

as shown

in Fig.

observed

with

to that

ligand.

In this

5 is an allosteric (Fritz,

with

of this

enzyme to catalyze

protein 1965).

Studies

designed

exhibited

to characterize

by the 2.

rettgeri

It

is

it and is

interesting

for

LDH's

J. N. 0.

R.,

and Vestling,

(1960)

J. Biol.

735

rabbit

by a number from

several

by fructose-1-6-diphosphate the nature

of the unusual

LDH are now in progress.

C. (1964) Chem.,

235,

J.

Biol. 810.

to

some

has been shown that activated

of protein

REFERENCES Anderson, S. R., Florini, 23'9, 2991. Dennis,-D., and Kaplan,

of

effectively

a requirement

In addition,

have been shown to be activated

a number

of an allosteric

unresolved.

regard,

2.

to a variety

as yet,

reflect

concen-

LDH exhibits

respect

indicative

might

in the

of the pyruvate

kinetics

is

LDH is,

to pyruvate

metabolites

of Streptococcus

behavior

rettgeri

of lactate

LDH isozyme

physiological

this

the

at low pyruvate

D(-)-specific

analogous

conditions. LDH from

rettgeri

have been shown to be allosteric

however,

physiological

is

these

in

to catalyze

rate,

function

The g.

difficulty

enzyme was assayed

reaction

to be a sigmoidal

DPN as the

as the acceptor.

under

ability

of

DPN as the

an L(+)-specific

When this

by DPNH, the

1965).

with

of the

reaction

also

reduction

in the case of the 2. speculate,

because

purified

and Kaplan,

a similalr

as yet,

reaction.

lactate

capacity

with

when APDPN serves

have recently

was shown

(Tarmy

the KM for

observed

reduced

to pyruvate

(1965)

which

of pyruvate

that

of the physiological

of the physiological

direction

the markedly

of lactate

been tested,

the reverse

Tarmy and Kaplan

for

conversion remains

is much higher

possibility

reverse

explanation

The possibility

cofactor This

a complete

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Chem.,

of species

Vol. 22, No. 6, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fritz, P. J. (1965) Science, 150, 364. Kaplan, N. O., and Ciotti, M. M. (1956) J. Biol. Chem., z, 823. Kline, L. and Barker, H. A. (1950) J. Bacterial., 60, 349. Lineweaver, H., and Burk, D. (1934) J. Am. Chem. Sot., 56, 658. Nisselbaum, J. S., Packer, D. E., and Bodansky, 0. (1964) J. Biol. Chem., 239, 2830. Tarmy, E. M., and Kaplan, N. 0. (1965) Biochem. Biophys. Res. Comm., 2l, 379. Wittenberger, C. L., and Flavin, M. (1962) J. Biol. Chem., 238, 2529. Wittenberger, C. L., and Haaf, A. S. (1964) J. Bacterial., 88, 896. Wolin, M. J. (1964) Science, 146, 775. Yoshida, A. (1965) Biochim. Biophys. Acta, 99, 66.

736