Analysis of the cold lability behavior of rabbit muscle phosphofructokinase

Vol. 66, No. 2, 1975

BIOCHEMICAL

ANALYSIS

AND BIOPHYSICAL

OF THE COLD LABILITY

RESEARCH COMMUNICATIONS

BEHAVIOR OF

RABBIT MUSCLE PHOSPHOFRUCTOKINASE

Paul

E. Bock,

Helen

R. Gilbert

and Carl

Frieden

Department of Biological Chemistry Division of Biology and Biomedical Sciences Washington University School of Medicine St. Louis, Missouri 63110 Received

July

24,1975

SUMMARY: Rabbit skeletal muscle phosphofructokinase has been previously shown to exhibit the characteristic of cold lability in phosphate buffers at pH values below pH 7 [Bock, P.E. and Frieden, C. (1974) Biochemistry 13, 4191-41961. Studies of the residual activity as a function of pH, reflecting the equilibrium between active and inactive forms of the enzyme, have been performed. These experiments show that the cold lability can be ascribed to a shift of the apparent pK describing the p&dependent inactivation to lower pH values at higher temperatures. The apparent pK, Hill interaction coefficient and heat of ionization for this process indicate that the equilibrium between the inactive and active forms of the enzyme may be controlled by the ionization of one or more histidine residues per enzyme subunit. In addition the apparent pK for the pH dependence of the residual activity at constant temperature is influenced by the presence of ligands which are substrates or effecters of the phosphofructokinase reaction.

It

is well

pH-dependent

(3)

dissociating

the

muscle

at pH values

in describing showed

to involve

phosphofructokinase below

and Hammes (4)

was a consequence to an inactive

enzyme

enough

rabbit

and Pavelich

inactivation

Frieden,

that

inactivation

and Frieden the

known

of the

form

the

characteristics

the

dissociation

for

enzyme of

process

of cold

(1,2).

active

that subunits

Bock and

(5)

lability

four

observed

that

at pH values

enzyme

to the

inactive

form. In this lability

paper,

we will

characteristics

Copyright o 1973 by Acadenfic Press. Inc. All rights of reproductiorl in atzy form reserved.

describe of the

results

enzyme. 564

which

a

Aaronson

quantitatively

two subunits.

this

of the

pH 7.2

showed

active

containing

a mechanism

about

undergoes

explain

the

cold

low

Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MATERIALS AND METHODS Phosphofructokinase:

Crystalline

obtained

as an ammonium

Chemical

Co.

bound

(lot

in these

by the

pm01 of fructose described

coupled

one enzyme

1,6-bisphosphate/min

were

performed (pH 8);

ATP; 2 mM fructose

6-phosphate;

units

25 units

of aldolase;

cl-glycerophosphate The reaction

in a final

buffer

against

measured

of

1

assay

by the

(pH 8), of

isomerase volume of

were

0.1

of the

100 volumes

mM NADH; 2 units

of

1 ml. enzyme

to the

above

and dissolved

They were

of

2 mM

and 2.5

centrifuged

mM EDTA.

conditions:

acetate;

4 mM NH4C1; 0.16

addition

enzymes

two changes

following

this

in

dialyzed

buffer

to remove

sulfate.

Enzymes, Residual

coenzymes

Activity

M sodium

phosphate

mg/ml

buffer

in 0.13

pH values

a thermostatted inactivation

water

were

obtained

treated

from

phosphate

stock buffers

mM dithioerythritol. held

at

the

bath

for

three

under

the

particular

565

solution

in

sufficient conditions.

(8-10

of known pH

Incubation

temperature

hours,

Sigma.

1 mM EDTA and 1 mM

a concentrated

M sodium

from

phosphofructokinase

(pH 8) containing

1 mM EDTA and 0.2

of different

were

Charcoal

was diluted

to 0.1

containing

and substrates

Experiments:

dithioerythritol

maximal

40 mM KCl;

was started

the

mM EDTA; 2 mM magnesium

dehydrogenase

M Tris-acetate

ammonium

0.01

triosphosphate

The auxilliary

mg/ml)

enzyme

formation

24" and pH 8 in the

of

mixture.

overnight

0.1

as the

at 24" and pH 8 under

33 mM Tris-acetate

0.1

of the

and cl-glycerophosphate

defined

at

to remove

1,6-bisphosphate

isomerase is

Sigma

of 160 unit&g

to fructose

unit

the

treated

Most preparations activity

was

below.

Assays

assay

(5).

triosephosphate

where

in 1 u&l ATP from

had a specific

by aldolase,

phosphofructokinase

The enzyme was charcoal

of NADH oxidation

dehydrogenase,

muscle

suspension

previously

experiments

rate

formation

sulfate

102C-8720).

ATP as described

used

rabbit

of the

mixtures experiment

time Aliquots

to obtain of the

in

Vol. 66, No. 2, 1975

incubation phate

BIOCHEMICAL

mixtures

buffer

(pH S),

described

above.

of

enzyme

active

the

specified

diluted

is

stable

ments,

the

data

to 5 &ml

1 mM EDTA, 0.2

The activity remaining

8 relative

presented to the

in

activity

reported

in these

temperature

of

incubation.

as

the

in the

incubation

mixture

at

Since

the

concentration range

(5). used

specific

enzyme

experiments

and assayed

way reflects

residual

of the

phos-

this

temperature

as the

pH values

the

dithioerythritol,

and enzyme the

RESEARCH COMMUNICATIONS

at O'C in 0.1 M sodium

at equilibrium

at pH 8 over is

ti

measured

pH, temperature

enzyme

below

were

AND BIOPHYSICAL

have

in these

activity

incubated

amount

experi-

at pH values

at pH 8.

been corrected

The

to the

RESULTS AND DISCUSSION The final tion

under

the

active

a function

given

residual

activity

conditions

and inactive

of

reflects

of pH (measured

the

1 shows the

as indicated

in Methods)

I

I

I

to undergo

equilibrium

Figure

forms.

r-

enzyme allowed

I

amount residual

inactivaof enzyme activity

at three

in as

different

,

100

P t5 F

U -l w 1

75

5c I-

t

i 25 ap

FIGURE 1. at 6', 15'

pH dependence and 25".

of

the

residual

activity

of phosphofructokinase

Rabbit muscle phosphofructokinase was incubated for 0.13 M sodium phosphate buffers containing 1 mM EDTA and erythritol at the pH and temperature shown and an enzyme 0.1 mglml. Enzymatic activity was measured as indicated

566

three hours in 0.2 mM dithioconcentration of in Methods.

Vol. 66, No. 2, 1975

BIOCHEMICAL

temperatures. is

As has been previously

pH dependent.

raised

the

(5).

similar

Thus,

not

that

this

Analysis activity plots

with

less

activity

an apparent of

the

shows

and positive temperature

carboxyl

groups

negative

or quite

of ionization

over

occur

consistent

30" yields

plots

Ionizable

lysine

the

of the

apparent

pK strongly

residues

is

responsible

be expected,

that

linear

Hill 4 and 5. curve

is relatively

the heats

with

decreases

of ionization

since

they

large

heats

groups.

at a given

the

ionization

pH dependent

of

are either

positive

amino

obtained that

residual

pK value

results

how-

the

ionization

apparent

data

temperatures.

activity

and N-terminal

the

and the

of between

of

acids

imply

for

period

residual

for

of ionization

some irrevers-

at physiological

these

amino

heat

cases

would

coefficient

with

apparent

the

beyond

Thus the

of the

histidine

at

pH dependent

The value value

described

equilibrated

It

heat

This

to enzyme

1 shows

The values

histidine,

pH values.

of pH yielding

apparent

is

at 6" and pH 6.6

hour

even

activity

temperature

lability

three

in Figure

increased.

small.

the

function

the

lower

in these

shown.

kcaljmole).

are not

include

However,

curves

that

is

1.

of the

(8-10

as the

temperature

interaction

shift

cold

residual

as the

towards

the

would

Hill

that

compared

cooperative

the

enzyme equilibrated

to those

the

RESEARCH COMMUNICATIONS

(1,2)

of

to occur

same process

temperature

large

example,

comparable

a highly

The direction with

for

of any of

is

shifts

characteristics

appears

therefore

ever,

curve

shown in Figure

inactivation

shown

is now observed

Increasing

same pH.

to those

ible are

the

show considerably and the

it

activity

explains

previously

25"

However,

residual

observation

will

AND BIOPHYSICAL

pH and of

behavior

of

enzyme. Studies

as a function be biphasic between

active

a two state

of the

time

of pH at at certain

course 6"

the

inactivation

(5) h ave shown the

pH values.

and inactive process.

of

This

result

enzyme may not

The equations

for

567

the

of

kinetics

phosphofructokinase of inactivation

implies

that

be strictly apparent

the

equilibrium

interpretable Hill

to

interaction

as

Vol. 66, No. 2, 1975

coefficient and in data

and heat some cases

of

of

give

the

rise

apparent

residues

particular fluence

At

a given

or effecters activity, dissociation 6-phosphate

behavior,

of that

on the

ionization

subunit.

While

shifts

the

residual

However,

the

temperature

chemical

of

one or more

modification

is not

clear

what

it

weight

the

changes

which effect

apparent

does

in-

(7).

pK value

curve

of

such modification

for

pH dependence

as shown in Figure activity

effect

are substrates the

the

to inactive

phosphofructokinase

also

Thus,

(6).

heart

the

process.

can be complex,

of sheep

ligands

influence

conditions

effect

temperature,

is

plots

dissociation

molecular

reaction

situations Hill

for

RESEARCH COMMUNICATIONS

the

by the

pH dependent

these

these

pK value

residues

allosteric

has on the

under

per enzyme

histidine

in

to non-linear

that

may be dominated

histidine

AND BIOPHYSICAL

of ionization

1 suggest

Figure

dependence enzyme

BIOCHEMICAL

for 2, the

towards

the

the

reaction

of the

residual

association-

substrate the

left

fructose(i.e.,

PH

FIGURE 2. The effect ence of the residual

of fructose 6-phosphate and citrate on the activity of phosphofructokinase at 6'.

pH depend-

Rabbit muscle phosphofructokinase was incubated at 6O for three hours in 0.13 M sodium phosphate buffers containing 1 ti EDTA and 0.2 ti dithioerythritol at the pH values shown and an enzyme concentration of 0.1 mg/ml. Residual activity in the presence of 100 I.IM F6P (-A-), no additions (-O-), and 5 IIN citrate (-0-).

568

Vol. 66, No. 2, 1975

lowers

the

curve

BIOCHEMICAL

apparent

towards

pK value)

the

right.

fructose

6-phosphate,

ATP (not

shown)

as both Residual

1,6-bisphosphate that of

the

This

a substrate activity

residual

lowers

somewhat

one mode of action.

while

citrate,

The activator also

is

AND BIOPHYSICAL

the

more might

in

of the

the

presence

or fructose-6-phosphate activity

is

not

pK value.

and appears in light reaction of mixtures are very

a simple

shifts

the

1,6-bisphosphate,

apparent

be expected

and inhibitor curves

an inhibitor,

fructose

complex

RESEARCH COMMUNICATIONS

function

The effect

to involve of

like

the

role

at pH values

more

of than

of ATP below

7.2.

of ATP and fructose complex of the

and indicate concentration

ligands.

Acknowledgements: Health Service

Grant

This research was supported AM-13332 and by Training

in part by U.S. Grant GM-01311.

Public

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Hofer, H.W. and Pette, D. (1968) Hoppe-Seyler's Z. Physiol. Chem. 349, 1105-1114 Paetkau, V. and Lardy, H.A. (1967) J. Biol. Chem. 242, 2035-2042 Aaronson, R.P. and Frieden, C. (1972) J. Biol. Chem. 247, 7502-7509 Pavelich, M.J. and Hammes, G.G. (1973) Biochemistry 12, 1408-1414 Bock, P.E. and Frieden, C. (1974) Biochemistry 13, 4191-4196 Neet, K.E. (1974) Biophys. Chem. 2, 102-115 Setlow, B. and Mansour, T.E. (1970) J. Biol. Chem. 245, 5524-5533

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