Allosteric and isosteric modifiers of NADH binding to cytoplasmic malic dehydrogenase

Vol. 53, No. 2, 1973

BIOCHEMICAL

ALLOSTERIC

AND BIOPHYSICAL

AND ISOSTERIC

MODIFIERS

RESEARCH COMMUNICATKINS

OF NADH BINDING

TO CYTOPLASMIC MALIC DEHYDROGENASE M. Cassman Section

of Biochemistry and Molecular Biology Department of Biological Sciences of California, Santa Barbara, California

University

Received

June 4,

93106

1973

SUMMARY: The binding of NADH to cytoplasmic malic dehydrogenase is shown to be affected by a number of added ligands. One class of ligands appear to be analogs of a substrate for the enzyme, &-malate. These alter the binding constant for NADH without affecting the cooperativity of binding. In contrast, fructose-1,6-diphosphate behaves as an allosteric inhibitor at low enzyme concentrations, apparently by shifting the monomer-dimer equilibrium of the protein to the cooperatively binding dimer. The significance of these results are discussed in terms of a proposed regulatory function for the enzyme. INTRODUCTION Malic

dehydrogenase

function,

through

eucaryotic

cells

the

also

have

to the mitochondrial

enzyme,

(S-MDH),

has not

in a number coupling

ies

in our

coupled

been

of intralaboratory

have

interactions

tory

functions.

Further

sible

allosteric

modifiers

--in vitro

effects

the role

flow

schemes,

@ I973

all

of the

studies

were

--in vivo

from

Inc. reserved.

beef

heart

In con-

malic

(5-7).

(8).

regulatory

stud-

of NADH to S-MDH,

presented

for

of

some regula-

to search

The relationship role

impli-

The existence

initiated

exist.

dehydro-

Recent

the enzyme may have

The results

modifiers

(l-4).

in some way involve

binding

protein

therefore

of NADH binding.

to a potential

by Academic Press, irz any form

that

catalyzing

S-MDH has been

of which

a cooperative

suggested

and isosteric

S-MDH was isolated

of reproduction

Rather,

However,

protein,

of the cytoplasmic

metabolism

demonstrated

MATERIALS

Copyright All rights

the cytoplasm

defined.

equilibrium

such cooperative

allosteric

in

mitochondrial

cycle.

distinct

and extra-mitochondrial

to a monomer-dimer

both

localized

with

acid

structurally

clearly

of metabolic

associated

the tricarboxylic

a second,

enzyme is

the

that

in

this

genase, cated

an enzyme normally

participation

same reaction;

trast

is

out

pos-

here

show of these

S-MDH is discussed.

AND METHODS and purified 666

by the method

of Guha,

BIOCHEMICAL

Vol. 53, No. 2, 1973

Englard

and Listowsky

isocitrate,

C grade,

1,6-diphosphate free

(9).

I&&-isocitrate

were

from Boehringer.

Bartlett.

Measurements

cent

of protein

quenching

of NADH-enzyme

volume

dilution

potassium

was 2.0 ml,

were

mostated

performed cell

were

following added

using

with

buffer,

fluorescence

All

the

Elmer

at ionic

were

carried

0.05.

were

for

out

The

corrected

emission.

an enzyme solution

(8).

MPF-IIA

strength

of the

The per-

saturation

titrations

10 ul aliquots

was

Grant

at 280 nm.

measurements

an Eppendorf

and allo-

made by following

fractional

non-linearity

which

of fructose

from Dr.

on a Hitachi-Perkin

phosphate

dependent

salts

of 3-phosphoglycerate

were

at 20-21'.

by equilibrating

block,

concentration

interactions

performed

and all

and concentration

tions

salt

was a gift

was linear

thermostated

trisodium

g-glucose-6-phosphate,

at 340 nm upon excitation

were

spectrophotofluorometer

and --D,L + -allo

The sodium

Glucose-1,6-diphosphate

fluorescence

in sodium

Calbiochem.

The barium

Sigma grade.

measurements

at pH 6.0

from

per mole NADH bound

Fluorescence

initial

obtained

RESEARCH COMMUNICATKJNS

A grade,

g-fructose-6-phosphate,

obtained

quenching

NADH and g-malate,

were

(FDP),

AND BIOPHYSICAL

for

The titra-

5 min.

in the

of NADH solutions

ther-

of known

micropipette.

RESULTS Titrations of added

NADH were

Two classes

of ligands

ligand.

identified.

The first These

-L-malate. ness

of S-MDH with

class

include

of allo-isocitrate

NADH binding isocitrate

all

D-malate,

with

added g,&

had no effect).

These

substituted

four-carbon

dicarboxylic

site

configuration

from

steric

presence creased modifiers

of members sigmoid appear

of this

character to alter

class compared the

of modifying

+ allo

of the natural

the observation material,

compounds acids,

while

can all with

of +nalate. generated

binding

to enzyme alone binding

667

and absence

NADH binding

and allo-isocitrate.

from

intrinsic

in the presence

to be analogs

citrate,

that

out

capable

appear

was inferred

curve

carried

of a change

a-hydroxyl

groups

Titrations

with

curves

constant

which 1). for

effectivein

the

allo-free

be represented

(Fig.

substrate, (The

added

were

D,&-

as a- or Bof the oppoNADH in the showed

However,

no inthese

NADH, resulting

in

Vol. 53, No. 2, 1973

BIOCHEMICAL

0

I

2

3

AND BIOPHYSICAL

4 TOTAL%ADH

6 7 ADDEDlM/LxlO’J

8

3

RESEARCH COMMUNICATIONS

IO

II

12

Figure 1. Titration curves of S-MDH with NADH, in the presence and absence of added ligand. The enzyme concentration was 5 ug/ml, and the ligands were added (o),+ D,&, f allo-isocitrate; at a concentration of 5x10e3M. (o), enzyme alone; (O), + g-malate; (A), + citrate; (A), + g-fructose-l,&diphosphate. The ordinate is fractional saturation of enzyme with respect to NADH. 7 = moles NADH bound per mole enzyme, and n = NADH binding sites per mole enzyme.

a binding tein

curve

displaced

in the

dependence

reported

range

relative lo-200

ug/ml

previously

Titrations

at pro-

the protein

concen-

to enzyme alone.

(8),

indicated

that

was unaltered

in the presence

of

ligands. The second

6-diphosphate

class

of effecters

@HP).

became distinctly lated

is

concentrations

tration these

which

At low enzyme

sigmoidal

intermediates

has as yet

in

such

diphosphate,fructose-6-phosphate, of

the Scatchard

showed

in

the presence

centrations

of enzyme

the monomer-dimer

alone

plots

NADH binding

obtained

The addition

of citrate

IJ-fructose-l,

the binding

curve

of FDP (Fig.

(Fig.

titration

produced

Other

formation

distinguished with a small

phosphory-

An

enzyme concentrations of high

the F'DP may act of the

NADH

no effect.

characteristic

the

effector

in a manner

con-

by displacing

cooperative

by following

the appropriate inhibition

668

is

for

glucose-1,6showed

at different

indicating

to favor

can be further upon

obtained

2),

1).

as glucose-6-phosphate,

of PDP the binding

equilibrium

The two classes

one member,

and 3-phosphoglycerate,

examination that

concentrations

the presence

of glycolysis,

only

dimer

(8).

changes

in

(Fig. reflecting

3). a

Vol. 53, No. 2, 1973

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

plots of S-MDH titrations with NADH at different Figure 2. Scatchard centrations, in the presence and absence of FDP. The solid lines are of enzyme alone. (o), 7 pg/ml ewzyme; (41, 70 pgfml enzyme; (A), 210 The broken lines are titrations in the presence of,lO-2M FDP. zyme. enzyme; (A), 70 pg/ml enzyme. and the ordinate is fractional The abscissa is fractional saturation, tion divided by the free NADH concentration.

0’ 0

I I

1 3

I 2 TOTAL

LIGAND

4 ADDED

5

enzyme contitrations pg/ml en(01, 7 ug/ml satura-

6

ImMi

Figure 3. Inhibition of NADH binding by added citrate and PDP, The . . enzyme concentration was 5 pg/ml, and the NADH concentration was 1.2x10-%. (VI f citrate titration; (o), PDP titration. The ordinate is fractional saturation of enzyme with respect to NADH.

669

Vol. 53, No. 2, 1973

normal

binding

showed

a marked

BIOCHEMICAL

isotherm.

In contrast,

cooperativity.

was approximately

AND BIOPHYSICAL

the

inhibition

of NADH binding

The concentration

1x10s5M

for

citrate,

RESEARCH COMMUNICATIONS

for

by FDP

half-maximal

and 4x1Cm3M for

inhibition

FDP.

DISCUSSION Many of the recent anisms

have followed

of their

controls.

properties

are

pathway,

advances

on the

Since

the observation

that

evaluation

not

appear that

that

phosphofructokinase inhibitors

limited e

under

levels

aerobic

that

shown by the 3), that

allows

maximal found

--in vivo.

--in vivo.

is This

However, approximately

the

is

brain

of FDP increased

a concentration

concentration one order

670

out (15).

These in

of cooperativity of added

FDP (Fig.

span consistent

of magnitude

on -in

2 to 5-fold

of FDP required

may be due to several

gly-

and glycolysis

carried

to S-MDH as a function over

and

accelerate

and heart

degree

at the

activators

inhibited

(14)

involve

has been

located

between

is

a re-

the

It

have been

The high

conditions.

to operate

discrepancy

rat

with

regulation.

suggests

PFK, and thus

the enzyme

concentration

of NADH binding

the FDP effect

strongly

studies in

together

in metabolic

in glycolysis

Parallel

steady-state

to anerobic

inhibition

while

However,

by FDP, required

The interplay

intermediates

inhibition

observed

point

there

capacity.

of glycolysis.

such as to stimulate

conditions.

the

aerobic

control

control

(PFK)(ll-13). is

enzyme

effector

to the

conditions,

of glycolytic

demonstrated from

enzyme

anaerobic

under

going

reaction

of this

colysis,

of the

catalyzed

conditions,

manner,

exhibited

nature

in a metabolic

reaction

of those

mech-

of regulatory

point

NADH in a cooperative properties

related

a major

existance

in any regulatory

of FDP as an allosteric

accepted

and the

enzymatic

either

functioned

significance

ment of S-MDH in phenomena generally

of the

allosteric

of the possible

enzymes

the possible

to satisfy

it

control

enzyme at some branch

S-MDH can bind

and striking

The existence

for

of the

of metabolic

of regulatory

irreversibility

to suspect

the specific

identification

the position

S-MDH did

was no reason

the understanding

Common indicators

and the effective

(10).

in

larger

factors.

with

for

half-

than

that

Observations

BIOCHEMICAL

Vol. 53, No. 2, 1973

with

a number

of preparations

very

labile.

This

Cassman, action

may modify

The results

dehydrogenase

NADH.

(16).

A possible

model

genase

cytoplasmic

for

of FDP would plasmic

inhibit

funnel

levels

reducing

S-MDH would

binding

equivalents function

active

site.

the degree

analogs,

Since it

substrate,

these

seems most and alter

interactions.

Of this

on NADH binding

of glycolytic controls

increased

a reoxidation Under

on S-MDH. dehydrolevels of cyto-

aerobic

conditions,

on NADH binding, via

as a secondary

observed

a modifier

and thus

S-MDH and the malate

control

with

which

citrate,

of cooperativity

centration.

the

the

of lactic

S-MDH and lactic

inhibition

that

point,

shuttle.

by responding

to the

activity.

i.e.,

In contrast,

the

re-

in regulating

by the

system.

the

investigated.

re-oxidation

conditions,

the mitchondria

interactions

modifier,

anaerobic

relax

into

of phosphofructokinase

allosteric

the

and

for

the control

may be provided

dehydrogenase

of E'DP would

therefore

a role

of NADH to S-MDH and allow

the lactic

The cooperative

effect

Under

being

that

between

(Vetterlein

The possibility

currently

for

a competition

NADH.

of NADH.

has suggested

to the enzyme

co-substrate

S-MDH may play

Such a link

of the FDP response

studies

the

is

and the mechanism

involve

NADH through

the lowered

level

would

that

Newsholme

in red muscle

NADH may be related

as well,

shared

relative

kinetic

binding

to FDP is

a characteristic

preparation,

cooperative

suggest

is

oxaloacetate,

PDP binding

presented

of cytoplasmic

that

the response

effectiveness

preliminary

indicate

NADH, can induce

the

the purified

In addition,

state.

oxaloacetate

flow

in

RESEARCH COMMUNICATIONS

that

effect

As a result,

altered

in progress),

with

of an allosteric

enzymes.

may be considerably in the native

of enzyme indicate

lability

by many allosteric

AND BIOPHYSICAL

binds

probable

group, appears

nor can all

that

act

the affinity

for only

too small

671

is

its

distinct

dependence

from

local

normally

affect

on protein

con-

to be significant.

as &-malate

at the

same site

electrostatic found

the

neither

be represented

by binding

NADH through

citrate

of an

and g-malate

compounds they

characteristic

at a site

allo-isocitrate,

of NADH binding, latter

FDP are

or steric

in cells, Thus,

as the

there

and its are no

Vol. 53, No. 2, 1973

strong

reasons

BIOCHEMICAL

to believe

that

AND BIOPHYSICAL

such isosteric

RESEARCH COMMUNICATIONS

effecters

are of physiological

im-

portance. This

work

was supported

by a University

of California

faculty

research

grant. REFERENCES 1. Delbriick, A., Schimassek, H., Bartsch, K., and Biicher, T., Biochem. Z., 331, 297 (1959). 2. Siegel, L., and Englard, S., Biochem. Biophys. Acta, 66, 101 (1962). 3. Thorne, C. J. R., and Cooper, P. M., Biochem. Biophys. Acta, 2, 397 (1963). 4. Kitto, G. B., and Kaplan, N. O., Biochemistry, 2, 3966 (1966). 5. Lardy, H. A., Shrago, E., Young, W., and Paetku, V., Science, I&, 564 (1964). 6. Krebs, H. A., Gascoyne, T., and Notton, B. M., Biochem. J., 105, 275 (1967). 7. Marco, R., and Sols, A., Fed. Eur. Biochem. Symposium, 2, 63 (1969). 8. Cassman, M., and King, R. C., Biochemistry, 11, 4937 (1972). 9. Guha, A., Englard, S., and Listowsky, I., J. Biol. Chem., 243, 609 (1968). 10. Atkinson, D. E., Ann. Rev. Biochem., 21, 85 (1966). 11. Newsholme, E. A., and Randle, P. J., Biochem. J., so, 655 (1961). 12. Passoneau, J. V., and Lowry, 0. H., Biochem. Biophys. Research Commun., L, 10 (1962). 13. Mansour, T. E., J. Biol. Chem., 238, 2285 (1963). 14, Lowry, 0. H., Passoneau, J. V., Hasselberger, F. X., and Schule, D. W., J. Biol. Chem., 239, 18 (1964). 15. Williamson, J. R., J. Biol. Chem., 241, 5026 (1966). 16. Newsholme, E. A., in Essays in Cell Metabolism (Ed. by Bartley, W., Kornberg, H. L., and Quayle, J. R.), p. 189 (Wiley-Interscience, London, 1970).

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