Substrate activation of pyruvate carboxylase by pyruvate

Substrate activation of pyruvate carboxylase by pyruvate

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Vol. 37, No. 5, 1969 SUBSTRATE ACTIVATION Helen OF PYRUVATE CARBOXYLASE BY PYRUVATE* Taylor, ...

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 5, 1969

SUBSTRATE ACTIVATION Helen

OF PYRUVATE CARBOXYLASE BY PYRUVATE*

Taylor,

Department

J.

Nielsen

of Biochemistry, South Australia,

ReceivedSeptember

and D.B.

Keech

University 5001.

of Adelaide,

12, 1969

SUMMARY

A kinetic analysis of pyruvate carboxylase isolated from sheep liver and using pyruvate as the variable substrate revealed non-Michaelis Menten kinetic. Double reciprocal plots were biof 222 were obtai.ned. Hill plots prepared phasic and R, values from the initial velocity data showed that at low pyruvate concentrations, the slope of the line varied from 1.0 to 0.7 and then With increasing pyruvate decreased to 0.4 at 0.8 x lo-3M-pyruvate. concentrations, the slope increased to a value of 1.0. This type of kinetic behaviour is typical of a system in which there is negative cooperativity with respect to ligand binding with concurrent progressive substrate activation. The fact vertebrate

the

that

trolled

by the

studies

reported liver

1966;

the

here

suggest there

of

to

this

for

(pyruvate:C02 is

Scrutton

activity

of

pyruvate in

and Utter, the

is a negative-type

activity

ligase

of

to the

of

(ADP), allosteric

1967)

enzyme -in

has led

vivo

is

con-

acetyl-CoA.

carboxylase

addition

the

a homotropic

concentration

using that,

essential

activation

intracellular

acetyl-CoA, pyruvate

the

et al.,

hypothesis

is

carboxylase

and that

(Barritt

sheep

acetyl-CoA

pyruvate

EC. 6.4.1.1.) effect

that

The

isolated control

cooperativity

from

exerted in

the

binding

enzyme.

EXPERIMENTAL Pyruvate

carboxylase

was purified

*This work was supported by Grant Research Grants Committee. 723

from

65/15780

freeze-dried

fro-m the

by

mito-

Australian

to

BIOCHEMICAL

Vol. 37, No. 5, 1969

chondria

isolated

involved

extraction

with

(NH4)2S04

The soluble

followed

of

Enzymic

activity

described (total

the

0.50

linear

for

fuging

to

Using

all

of the

remove

( 1" x l"),

(in dried

using

the

(1964).

seccnd

addition

c.p.m.

of

were

protein, to Whatman

and counted

in

method

1.25,

ATP; 4,

kmole);

reaction

0.13, at

After

of

the

centri-

squares

scintillation correct

was

supernatant

3MM filter-paper

to

30° for

velocity

used.

a Packard

mixture

2,4-dinitrophenyl-

aliquots

triplicate)

method

incubated

saturated the

gradient.

6C, tris-HCl

per

concentrations

ratio

pH 7.5.

assay

as required;

conditions

precipitated

at

of the

micromoles/ml;

mixtures

pyruvate

glycol.

on DEAE-

The standard

in

Reaction

channels

twice

and the

(6 x lo5

these

polyethylene

by a KC1 concentration

varied

by the

procedure precipitation

by a modification

bicarbonate

HCl.

applied

pH 6.5

cc,ntained

and enzyme.

hydrazine

at

--et al. ml)

and stopped

meter

first

tris-pyruvate,

sodium

acetyl-CoA 4 min.

the

with

chromatographed

was measured

pH 8.4; 5,

material,

enzyme was effected

volume

W12;

freeze-dried

by precipitation

by Gailiusis

buffer,

The unpublished

was then

columns,

Elution

the

RESEARCH COMMUNICATIONS

liver.

sheep

of

protein

cellulose

were

from

AND BIOPHYSICAL

spectro-

for

quenching.

RESULTS AND DISCUSSION When initial function

of

pyruvate

rectangular

hyperbola

reciprocal biphasic

plots

exceeded

Michaelis-Menten for

negative

the

value

from

the

81 which

hyperbola.

These binding

the

data

of

for

provide

of pyruvate

1).

the

velocity)

expected

Double

the

a pyruvate of

222,

a classical

strong to

from

exhibited

ratio

saturation is

724

(Fig.

experiments

(i.e.,

as a

deviation

observed

these

Rs value

and 10% of

and plotted

a pronounced

of

cooperative

measured

was consistently

and the at 9%

were

concentration,

obtained

effect

concentration far

velocities

evidence

enzyme

a

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 5, 1969

and Koshland,

(Levitzki

initial It

velocity

1969).

data

can be seen that

presented the

in

slope

At very

concentration.

A Hill

low

Fig.

of the levels

plot

from

1 is presented

line

of

prepared

varies

with

pyruvate,

the

the

in Fig.

2.

pyruvate slope

varies

3

0

. 1

1 3

5

$103 2

6

cone”.

Pyruvate

Velocity plotted as a function of pyruvate Fig. 1. concentration, The points marked are experimental values. Inset: reciprocal of velocity plotted as a function of the reciprocal of the pyruvate concentration. The curves were drawn from the kinetic constants obtai.ned by fitting the data for separate parts of the biphasic plots to a computer program for a rectangular hyperbola using the method of least squares (Wilkinson, 1961). These values were; apparent Km 1.5 x lo-4M and 6.2 x 10-4Mo

from

1.0

to

transition pyruvate) tions.

0.7 from

and then low

and then This

type

decreases

to high

enzymic

increases of plot

Levitzki

and Koshland

negative

cooperativity

to is

(1969) with

to 0.4

1.0

region

(i.e.

at high

pyruvate

to that

f or a system

725

the

activity

identical

respect

in

to

in

binding

1.0

of x 10B3M-

concentra-

predicted which but

there positive

by is

BIOCHEMICAL

Vol. 37, No. 5, 1969

cooperativity

with

respect

An alternate enzyme preparation

AND BIOPHYSICAL

to

RESEARCH COMMUNICATIONS

catalysis.

explanation

for

there

two enzymes,

exist

the

above

data

is

that

in

one functional

the

at

+ >: > 11 0 -I

1 0.5

I

I 1.0 LOG

I l-5

~YRuvATE]

D

+ 1

Fig. 2. Hill plot for the data presented in Fig. The points marked are experimental values and the curves were drawn as described in Fig. 1.

1.

low

and the

other

However,

against

activation

is

ure.

Also,

this present

the

.and V max values procedure

The effect

varied

from

pH 7.5

stages

and the

the

that

ccnstant

fact

of the ratios

the

substrate

purification of the

throughout

certain

and shown

the

parameters

to have

and 9.0,

(pH 8.4).

very

For it

was more pronounced optimum

concentrations.

is

characteristics.

between

the

all

of varying

kinetic

cooperativity

at

pyruvate

two

procedapparent

Km

purification

1).

was examined

relative

at high

explanation

Rs values remain

(Table

mixture

functional

However, 726

little

example,

appeared as the

of

that

the

effect

on the

when the

pH was

the

negative

pH was shifted

when these

reaction

curves

away were

normal

Vol. 37, No. 5, 1969

ised

they

BIOCHEMICAL

were

found

to

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

be very

similar

to

each other.

Similar

TABLE 1 COMPARISON OF THE KINETIC

PROPERTIES AT VARIOUS STAGES OF

THE PURIFICATION

Ratio Stage of Purification

Rs value

of the two

Kmrs

were

a ten-fold

range

carboxylase Williamson cellular

it is

1.4

276

5.91

1.69

0.93

3.5

294

6.32

1.62

1.11

5.1

222

5.94

1.88

0.84

6.6

exerts

the

the

on the

cooperativity substrate

either

accepted

that

by the

with

here

indicates

respect

over

it the

that

at

concentration enzymic to

the

appears 727

was examined

acetyl-CoA the level

of

that

from

acetyl-CoA, the

known

seems unlikely

least

of

to be the

for that

activity. with

can have

activity. binding

of pyruvate

Km values

enzymic

the

The sheep

a profound

Although

negative

pyruvate

with

most

over

or MgATP2-.

activity

apparent

enzymes,

control

of

out

and the

a fine

activation

cellular

h ave pointed

pyruvate

velocity

of

vertebrate

level

reaction

levels

(1968)

presented enzyme,

when the

of acetyl-CoA

acetyl-CoA

quent

0.94

is widely

et al.

for

influence

1.74

controlled

acetyl-CoA

liver

5.65

of fixed

levels

evidence

239

obtained

Although

'max's

Spec. Act.

mM

Polyethylene glycol step DEAE-cellulose column pH 6.5 DEAE-cellulose column pH 7.5 G200 Sephadex column

results

Inflection point of double reciprocal plot

likely

subseexplana-

Vol. 37, No. 5, 1969

tion

the

for

independent

BIOCHEMICAL

observed pyruvate

catalytic

kinetics,

is

sites

not

significance

of these

results

The apparent

K m value

for

1.5

x 10q4M which

is

reported

physiological

(Ballard

and Hanson,

that

activity

the

curve the

would liver

of

respect 1967),

MgATP2-

cooperativity

affected in

the

by the enzyme

only

(Barritt

respect

the

exhibits et al.,

to

functional explanation.

activity higher

the

1968).

range than

liver

Thus it

would

portion

is

the

ruminant

when a large

complexity positive 1966;

and Mg 2+' (K eech and Barritt, with

the

and

seem

of the

velocity

influx

into

occurred.

emphasises which

physical

lower

upper

separate

by the

in

al -et --7

precursors

carboxylase

to acetyl-CoA

not

three-fold

Baird

by the

different

of pyruvate

represented

of two

However,

pyruvate

1968;

be used

with

approximately

communication

pyruvate

is

RESEARCH COMMUNICATIONS

existence

excluded.

levels

of pyruvate

This

the

binding

properties

AND BIOPHYSICAL

of the

control

cooperativity Scrutton 1967),

with

and Utter, and negative

pyruvate.

REFERENCES Baird,

G.D., Hibbit, K.G., Hunter, G.D., Lund, P., Stubbs, M. and Krebs, H.A., Biochem. J., 107, 683 (1968). Ballard, F.J. and Hanson, R.W., Biochem. Biophys. Res. Comm., 3C, 100 (1968). Barritt, G.J., Keech, D.B. and Ling, A.-M., Biochem. Biophys. Res. Comm., 24, 476 (1966). Gailiusis, J., Rsne, R.W. and Benedict, C.R., Biochim. Biophys. Acta, 92, 595 (1964). Keech, D.B. and Barritt, G.J., J. Biol. Chem., 242, 1983 (1967). Levitzki, A. and Koshland, D.E., Jr., Proc. Natl. Acad. Sci., 62, 1121 (1969). Scrutton, M.C. and Utter, M.F., J. Biol. Chem., 242, 1723 (1967). Wilkinson, G.N., Biochem. J., 80, 234 (1961). Williamson, J.R. Browning, E.T. and Olson, M.S., Adv. in Enzyme Reg., 5, 67 11968).

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