AMP-activation of an allosteric NAD -dependent glutamate dehydrogenase

Vol. 28, No. 1, 1967

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

AMP-ACTIVATION OF AN ALLOSTERIC NAD -DEPENDENT GLUTAMATE DEHYDROGENASE H. B. L&John Department

of Microbiology, Winnipeg,

University Manitoba

of Manitoba

ReceivedJune 5, 1967 In most glutamate Lata,

bacteria,

higher

dehydrogenase

1964).

This

is

glutamate

genases for

NAD (L&John,

for

Neurospora

of

catalyzing

the

for

becomes

with

reason distinct

same reaction

(Sanwal

and

either

NADP

case

of beef-

the

facultative dehydro-

NADP, and another reported

yeast

(Holzer,

1957)

1967).

In all

dehydrogenase very

why there

cases

has been

important

coenzyme which

one

has been

1961),

glutamate It

only

two glutamate

and Neiderpreum,

physiological

enzymes,

in

situation

and Lata,

to be inducible.

these

However,

A similar

NAD-dependent

the

well-studied

one specific

1967).

(Dennen

the

determine

the

for

novellus,

found,

(Sanwal

Schizophyllum

shown

as in

Thiobacillus

have been

studied,

known to be present

dehydrogenase.

chemoautotroph,

and animals,

enzyme may be specific

or NAD or non-specific liver

plants

then

should

to

be two

specificities,

one could

conceivably

achieve. The enzymes

isolated

from

amenable what

to --in vitro studies has been suspected for

dependent

enzyme

communication effector

acts

reports

of the

in the

2. that

novellus may provide

some time,

a purely influence

NAD-dependent

viz.,

catabolic

especially

an answer that manner.

the

to NAD-

This

of AMP as an allosteric

glutamate

96

were

dehydrogenase

altering

Vol. 28, No. 1, 1967

the

BIOCHEMICAL

equilibrium

constants

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

so as to favour

The effect

is quite

specific

for

nucleotides

tested.

ATP,

apparently,

glutamate

breakdown.

AMP among a variety reverses

of

the

AMP

effect. MATERIALS AND METHODS The NAD- and NADP- dependent purified

from

Thiobacillus for

cultures

derived

novellus

induction

glutamate from

a single

(ATCC 8093).

of the

dehydrogenases cell

Growth

NAD-dependent

isolate

and other

enzyme will

were of

conditions

be published

separately. Purification

of Glutamate

harvested

Dehydrogenase.

by centrifugation,

Bacteria

washed

twice

pH 8, and resuspended in 5 vol containing 10 -3 M GSH (TG) buffer.

0.05

HCl,

included

at all

stages

concentration. sonic

The cells

disintegrator

ml)

at

solution at 37OC for

and held 5OC.

30 min,

at that

Tris-HCl,

using Further with

a linear

eluted

off

gradient

sulphate

debris

then

a column with

as separate 0.05

was removed

RNAse (5Opg/ within

2 vol

x g for out

The super-

rapidly

was made to

siphoned

of

a 10 KC Raytheon

to 55'C

5 min,

at 48,100

onto

AMP was at 10 -4 M

DNAse (50pg/ml),

for

pH 8,

15 min at 2OC.

brought

equilibrated

purification ammonium

with

extract

was carefully

30 cm) previously were

x g for

rapidly

viscous

and adsorbed

enzymes

Cell

pH 8, centrifuged

The supernate proteins

30 min at 5OC.

temperature

The thick

procedure in

was treated

M Tris-

M Tris-HCl,

disrupted

48,100

0.01

When necessary,

isolation

were

for

by centrifugation natant

of the

in

were

from

97

to

0.05

M

20 min at 2°C. the

denatured

of DEAE-cellulose

(2.5

0.05

pH 7.5.

M TG buffer,

peaks,

with

by differential

(45% saturation

cooled with

for

x The

some overlap,

M - 0.5 M TG, pH 7.5

was achieved

2 min

buffer,

precipitation

NAD-dependent

enzyme;

Vol. 28, No. 1, 1967

8lOCHEMlCAL

and 65% saturation ography Both

for

were

Enzyme Assays. was used enzymes

for were

A Gilford

Model

according

the

and rechromat-

same gradient

2000 recording

to

at pH 8.

the

spectrophotometer

at 340 rnp.

procedure

freezing

enzyme was stored

of in

and

desensitized

and thawing.

48 hours

The

of Sanwal

enzyme was easily

within

and used only

enzyme)

measurements

by repeated conducted

RESEARCH COMMUNICATIONS

250-fold.

The NAD-dependent

were

thawed

over

assayed

or alternatively, -2O"C,

the

purified

velocity

(1964).

assays

using

initial

to AMP activation all

NADP-dependent

on DEAE-cellulose

enzymes

Lata,

the

AND BIOPHYSICAL

Consequently,

enzyme purification,

suitable

aliquots

at

once when required.

EXPERIMENTS AND DISCUSSION The NAD-dependent the

reversible

Normal

formation

Michaelis-Menten the

and NADP-dependent of

a-ketoglutarate

kinetics

constants

of

NAD-dependent

glutamate

(see Km values

enzymes from

indicated enzyme

in Table

I)

both

that

favoured in vitro,

catalyzed

glutamate. the the in

equilibrium formation

the

of

absence

TABLE I Michaelis-constants with L-glutamate, and ammonia in

of NAD-dependent.glutamate dehydrogenase a-ketoglutarate, oxidized and reduced NAD, the presence and absence of AMP

Substrate

Km values

L-Glutamate

1.1

x 10 -3

2.5

x 10 -3

NAD

1.4

x 1o-4

2.0

x 1o-4

NADH a-Ketoglutarate

8.0 1.0

x 1O-5 x 1o-2

4.4 4.0

x 1o-6 x 10 -4

Ammonia

2.3 x 1O-2

5.0

x 1o-3

* AMP at a fixed

in moles/litre (- AMP) t+ AMP)"

concentration

of

98

10 -3 M .

Vol. 28, No. 1, 1967

of AMP.

BIOCHEMICAL AND BIOPHYSICAL

AMP modified

breakdown. were

the

The reaction

unaffected

kinetics

by AMP.

be restricted

Km values

RESEARCH COMMUNICATIONS

so as to favour of the

Consequently,

to the NAD-dependent

glutamate

NADP-dependent

data

in

this

enzyme

report

will

enzyme.

40.

l : (+I AMP o: (-IAMP

,

Fig.

l(a).

GLUTAMATE

- mM

NAD - mM

Effects

of AMP on the

glutamate

dehydrogenase

glutamate

to a-ketoglutarate.

0.2

M Tris-HCl,

concentration the

during

pH 9.5; of

same as for

0.05 (a)

kinetics

of NAD-dependent

oxidative

deamination

The reaction 1 mM AMP where

mM NAD. except

that

mixture

indicated;

Glutamate

contained and a fixed

The reaction

(b) .

of

mixture

was fixed

was

at 16 mM

and NAD varied.

When standard the

reaction

NAD-dependent

clearly (Fig.

sigmoidal la

synthesis modified

velocity

enzyme, in

the

it

and hyperbolic

(Fig.

2 a & b).

non-physiological

hyperbolic

substrate

was observed

direction

& b),

into

vs.

that

of glutamate in the

The sigmoidal

direction curve

plots

when extremely

concentrations

of substrates

99

plots the

were made on curves

breakdown of glutamate could high, were

be and used

were

Vol. 28, No. 1, 1967

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

r-1

Q - KETOGLUTARATE 0.4

1310

- mM 0.8

1.0

-I

a:+ AMP o:-AMP

0

0

2

4

6

8

a-KETOGLUTARATE 1-t

100

2

- mM

I

4 NADH

.,

6

8

x IO~mM

Fig. 2(a). Effect of AMP on the kinetics of NAD-dependent glutamate dehydrogenase during the reductive amination of a-ketoglutarate. Reaction mixture contained 0.2 M Tris-HCl, pH 8.0; 0.02 mM NADH; 40 mM (NH4)2S0 ; and 1 mM AMP where indicated. a-ketoglutarate as varie A substrate. (b). The reaction mixture was the same as for (a) except that cr-ketoglutarate was fixed at 1.67 mM and NADH was varied.

(25 - 50 mM glutamate, AMP-Effect. that

Attempts

could

curves

convert

the

mM NAD).

then

made to

observed

find

sigmoidal

a suitable

plots

concentrations.

into

AMP and to a lesser

The activation change

from

effector

is

effect sigmoidal shown in

reaction

AMP was inhibitory

extent,

ADP, were

was optimal to hyperbolic Fig.

1 a & b.

of glutamate (Fig.

hyperbolic

IDP,

curves

active

with

2 a & b) with

IMP)

and

as effecters. M.

The

AMP as

When AMP was used from

of

and protamine)

at 10m3 M - lO-4

formation

100

effector

Among a variety

(ATP, ADP, AMP, GTP, GDP, GMP, ITP, . . (poly-lysine, spermidine, polyadenine,

macroions

reverse

were

at physiological

nucleotides

only

and 0.2

in the

a-ketoglutarate,

a correspondingly

Vol. 28, No. 1, 1967

large

increase

ATP-Effect. act

BIOCHEMICAL

in

in Km for It

the

manner

experiment

opposing

manner

Fig.

ATP slightly

3.

are

a-ketoglutarate low

A theory

the

is that

regul .ation.

the

Further

involved

to suppose

in which

formation

to inhibit

work

the

plots

the

When tested

being

in

breakdown

done

an

of

on this

from

same reaction

the

at

reverse

of glutamate.

of ATP/AMP may be critical is

in

of glutamate

ADP and AMP inhibited

ratio

The results

Lineweaver-Burke the

I).

ATP may

ATP, ADP, and AMP act

the

activated

(Table

that

to AMP as an effector.

concentrations. ATP failed

RESEARCH COMMUNICATIONS

substrates

thesis

shown in

while

substrate

direction,

all

was a logical

opposite

of a typical

AND BIOPHYSICAL

in

this

aspect.

eo-

IS-

?

> IOot ATP a: AMP or ADP I: CONTROL

Fig. 3. function effecters. 6.67 mM and AMP

Rate of reductive amination of a-ketoglutarate as a of NADH concentration with ATP, ADP, and AMP as Reaction mixture contained 0.2M Tris-HCl, pH 8.0; a-ketoglutarate; 40 mM (NH412S04; and one of ATP, ADP at a concentration of 1 mM. NADH as varied substrate.

These regulation activity functions

investigations of

point

the NAD-dependent

to a probable glutamate

by AMP and ATP in T. novellus. in a purely

catabolic

manner

101

metabolic

dehydrogenase This supplying

enzyme,

seemingly,

ATP during

the

Vol. 28, No. 1, 1967

breakdown would the

BIOCHEMICAL

enzyme

the

necessary

to a state

glutamate.

A delicate activity

of

concept

that

catabolic

this the

becomes

may prove

that

are

Indirect

comes from induced sole

studies

when the carbon

involved

in

which

energy

the

supporting

is

the in

a largely

shown that

forced

source

sophisticated

equilibrium

regulate

have

organism

of

accumulation.

may function

and energy

to be a very

then

evidence enzyme

the

glutamate

could

activate

breakdown

of ATP accumulate,

NAD-dependent

as its

This

continuous

so as to favour

enzyme.

AMP and ADP

(ADP, AMP) that

of ATP/AMP ratio

manner

glutamate

levels

slightly

balance

ensures

RESEARCH COMMUNICATIONS

of ATP into

effecters

that

When high

may be shifted

enzyme

Breakdown

of glutamate.

provide

AND BIOPHYSICAL

to

rely

(L&John

regulation

this on

1967). for

enzymes

synthesis. ACKNOWLEDGMENT

Supported Canada.

by a grant The author

B. D. Sanwal

for

from

the

extends

many helpful

National

thanks

Research

to Drs.

discussions

I. of

Council Suzuki

the

of and

problem.

REFERENCES Dennen, Holzer, LGJohn, Sanwal, Sanwal,

J. Bacterial., 93, 904 D. W., and Neiderpruem, D. J. (1967) H., and Schneider, S., Biochem. Z., 329, 361 (1967) H. B., In Press (1967) B. D., and Lata, M., Canad. J. Microbial. 1, 319 (1961) B. D., and Lata, M., Modern Methods of Plant Analysis, Vol. VII, p 290, Springer Verlag, Berlin (1964)

102