Rat liver 6-phosphogluconolactonase: A low Km enzyme

Vol.71,No.4,

1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RAT LIVER 6-PHOSPHOGLIJCONOLACTONASE: A LOW Km ENZYME P.J. Instituto

Schofield

de Enzimologia

de la Universidad

Received

and A. Sols

de1 CSIC, Autonoma,

Facultad

de Medicina

Madrid-34,

Spain

June 28,1976

SUMMARY. By specific enzymic synthesis of the substrate 6-phosphogluconolactone ti bLtu, the Km for rat liver 6-phosphogluconolactonase was found to be 80 PM. This value is approximately 100 fold lower than the previously determined value, and is compatible with the kinetic parameters of both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and hence with the flux through the oxidative segment of the pentose phosphate pathway. The initial, involves that

catalytic

be modulated

aX.

(1)

in the

specific

in a completely

activities

activity

of the G6PDH at free

been

found

activity

activity

the pathway 6PG, viz,

site.

the

the

free

by the

action

acid

is

generally

of a specific

specific

(6PGL), assumed

6-phosphogluconolactonase.

Sapag-Hagar

is an apparent

imbalance

of conditions,

than

the

activators

corresponding

of G6PDH is

6PGDH/G6PDH

of flux

through

the lactone

hydrolysis

by spontaneous

range of 6PGDH have

or calculated

and the

to occur

dehydrogenases

However,

of an impairment product

be expected

in the physiological

low measured

The irnnediate

thus

a variety

much smaller

possibility

pathway

NADP-specific

there

liver,

occurring,

6-phosphoglucono-6-lactone

to the

rat

might

manner.

NADP+/NADPH ratios

results),

may suggest at that

for

of 6PGDH is

no naturally

(unpublished ratio

these

phosphate

It

of the two enzymesunder

the specific

Since

of both synchronous

that,

and that

- 0.01).

of the pentose

G6PDH* and 6PGDH.

activity

have demonstrated

(0.1

phase

two dehydrogenases,

the maximal

would ti

the

and oxidative,

of the hydrolysis

However,

little

*Abbreviations: G6PDH, glucose 6-phosphate dehydrogenase (EC 1.1.1.49); 6PGDH, 6-phosphogluconate dehydrogenase (EC 1.1.1.44); G6P, glucose 6phosphate; 6PG, 6-phosphogluconate; 6PGL, 6-phosphoglucono-b-lactone.

Copyrighr 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

1313

of lactone and/or is

Vol.71,No.4,

1976

known about lactonase

BIOCHEMICAL

this

lactonase

provides

in mamnalian

the link

between

6-phosphogluconolactonase, lactone

of approx.

In view

of the

This

the

communication

of the

Km value reports

RESEARCH COMMUNICATIONS

despite

the fact

two dehydrogenases.

&.

7 mM, and a total specific

systems

the

Kagawa ti

respective

dehydrogenases,

AND BIOPHYSICAL

(2)

have obtained

activity

of approx.

activities

of 7 mM for

the

For rat

liver

a Km value

for

35 U/g tissue

and Km values the lactonase

a re-investigation

that

the

(3).

of the two

seems anomalously

of the basic

kinetic

high.

parameters

lactonase.

Experimental. The livers of adult rats fed ud lib were homogenised with 4 vols of 5 mM K2HP0~-1 mM dithioerythritol-150 mM KCl-1 NM MgC12-0.5 reel CaC12, adjusted to pH 7.0. The supematant resulting from centrifugation at 30,000 g x 30 min at 4O was used itnnediately or stored overnight in a frozen state. For kinetic studies the reaction mixture contained, in a total vol of 1 ml, 5 mM NADP+, 100 mM imidazole pH 7.0, approx. 10 U G6PDH and 5 mM MgC12. The reaction giving 6PGL formation was started by the addition of G6P (cont. range: 30 - 200 pM) and after 30 set, approx. 2.5 U 6PGDH was added. After a further 30 set, tissue extract as a source of 6-phosphogluconolactonase was added. The change in extinction at 340 nm was recorded (path length: 1 cm). For measurement of the spontaneous hydrolysis of 6PGL at 25' and and pH 7.8 by the method of Horecker and Smyrniotis (4), the reaction contained, in a total vol of 1 ml, 1.5 mM NADP+, 50 mM Tris (pH 7.0 or approx. 5 U G6PDH, 1 U 6PGDH and 5 mM MgCl*. The reaction was started addition of 50 pM G6P, and the extinction at 340 nm determined. Values are quoted as the mean f S.E.M. computed by the method of least squares. Results

and discussion.

Major

the unavailability

and lability

method

and Smyrniotis

of Horecker

enzyme studies.

In our

low and unreliable

yields

G6P which

is similar

The major

product

experimental only

in btiu amount

by the

(4) this

proved

method,

of 6PGL, despite obtained

was 6PG, the yield

method

in the

of the substrate.

to the value

manipulations

feasible

hands,

difficulties

The lines

to routinely

use of the appropriate

of G6PDH, and at a relatively

u&z,

of the

Preparation

bromine

by Horecker being proved the

lactone

1314

were

lactonase

routine

oxidation

are

kinetic

of G6P, gave

80% utilisation and Smymiotis less

than

of (4).

20%, and

unsuccessful.

Thus the

was to synthesise

mass of G6P and a relatively low pH to reduce

fit

of 6PGL by the for

approximately

the yield

synthesise

study

unsatisfactory

of lactone

to improve

of best

pH 7 mixture 7.8), by the

spontaneous

large hydrolysis

it

BIOCHEMICAL

Vol. 71, No. 4,1976

of the subsequent varied,

ranging

lactone.

Smyrniotis

(4),

presence follow

The values

from approx.

and 24 min at pH 6.4

(6).

By application

in which

order

G6P is

kinetics,

of the

&

with

hydrolysis

the half-life (4)

of 6PGL are

to 20 min at pH 7.0

a large

of the

half-life

Thus,

lactone

for

of the methodology

reacted

the

7 and 3.5 min respectively. preparation

cited

1.5 min at pH 7.4

of 6PGDH, spontaneous first

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

at pH 7.0

at room temp.

A&!& proved

of Horecker

excess

lactone

(5) and

of G6PDH in

at 25'

was found

and pH 7.8 being

and at pH 7.0,

the to

approx.

the

feasible.

8

.t 6 E

Fig.1. The rate of spontaneous hydrolysis of the 6-phosphoglucono-&lactone formed &I b.Ltu at varying concentrations of lactone. The values are expressed The regression line was computed by the method as the mean * S.E.M. (n = 3). of least squares (r = 0.96, p
In the presence (cont.

range:

GPGDH after lactone This giving half-life

of a large

30 - 200 uM) was rapidly 30 set

demonstrated

at pH 7.0 was 5.5 rate

excess

of spontaneous

a half-life

that

of G6PDH, 69.8 converted

the

+ 0.3% per min over hydrolysis

exhibited

of 12 min at pH 7.0,

as determined

rate

by the method

which described

1315

+ 1.4% of the G6P

to the

lactone.

of spontaneous the first is

hydrolysis

concentration order

kinetics

in good agreement above.

Addition

Subsequent

range

of of the

used.

(Fig.1) with addition

the

Vol.71,No.4,

BIOCHEMICAL

1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3 3 0.2 _ ; 9

I

Q 0.1 -: I I I

I 1 TIME (min)

I 0.5

L 2

Typical 6-phosphogluconolactonase assay. The reaction mixture Fig.2. contained 5 mM NADP, 100 IIM imidazole pH 7.0, approx. 10 U GLPDH and 5 mM MgC12. The reaction was started by the addition of G6P (65 PM). After 30 set approx. 2.5 U 6PGDH was added (A) to determine the rate of spontaneous hydrolysis of the lactone. After a further 30 set tissue extract (10 ul) was added (B) and the initial velocity of the lactonase determined.

of tissue

extract

absorption

of the 6PG formed

was routinely

used for

Fresh the

extract

only

extracts

as manifested

thus

normal to that

of magnitude

less

be noted

by the non-specific

in the millimolar

range;

that

in turn,

lactonase

resulted

showed

increase

in

was due to

(Fig.2).

animal

values

was quite in less

less

the

lactone

than

for low.

than

variability

This

method

Kagawa oxamate the

1316

determined et uL.

method

(2)

pH of the

of the

in

to be 80 yM, with (Fig.3).

The total but

by Kagawa ti measured

and this

Km value

Storage

10% loss

by Kawada et (Le. (3), that

the

of the Km value.

was found

of 10 U/g tissue

found

that

erratic

the value

state

similar

should

to give

the Km for

in the

of NADP+ which,

that

extract

conditions,

activity

it

thawed

in the

studies.

tended

in a frozen

two orders

of lactone

kinetic

overnight

is

However,

the

but suggested

these

activity is

tissue

and the

maximal

hydrolysis

by the endogenous

lactone,

activity, Under

rapid

at 340 nm due to reduction

oxidation

for

caused

method

incubation

the Km value a&

(2).

the disappearance is sensitive medium was not

BIOCHEMICAL

Vol. 71, No. 4, 1976

/ / / / /

AND BIOPHYSICAL

I .Ol

I .02

RESEARCH COMMUNICATIONS

I .03

'%PGL

(pM I-'

Fig.3. Lineweaver-Burk plot with 6-phosphogluconolactone as the substrate synthesised &I &Au. The reciprocal of the initial velocity is expressed as (pmole/min)-'. The regression line was computed by the method of least squares (r = 0.99, p
specified,

and that

lineality that

of the

the

mental

high

the

reaction

Km value

artifact

at 61'

reduction

in lactonase

for

above,

we have

5 min caused

30% loss

loss

obtained

to keep the

(2)

Hence, is

and relatively

of rat

the oxamate

liver

With that

heat whereas

insensitive

method

method,

found

resulted

for

that

of "heat

in a marked

of analysis

of the

treatment

appear

of experi-

method

treatment

would

a result

supernatant

the specific

it

exact

extract

out-

at 60'

5 min at 65'

for

caused

activity. within

at higher

the

importance,

from G6P by G6PDH is of the

physiological

pH values

lactone

oxidation

was difficult the assay".

using

in activity,

of lactonase

since

(3),

determined

much greater

the

during

activity".

assumes

as is

"it

by Kagawa et &.

3 minutes

The use of pH 7.0, compromise,

velocity

Kawada et uX.

treatment

complete

stated

due to the non-specific

determination.

lined

authors

the spontaneous

while hindered

free

range,

at lower by the

6PG by 6PGDH.

1317

hydrolysis

pH values

displacement

is a satisfactory

the

of lactone formation

of

of the equilibrium,

The determined

K,,, value

of

BIOCHEMICAL

Vol.71,No.4,1976

80 UM for closely

the

the kinetic

previously for

lactone,

the

and the total parameters

determined

Km value

phosphate

activity

in the

range

since

both

Hence,

are

of the

same orders

is

presumably

not

that

the

&I uivo,

of 7 mM.

hexose

conditions.

lactonase

itself

whereas

was truly

the

would

total

of 10 U/g, than

in the range

may prevail

30-70

the

PM, and

lactonase

and the

of NADPH, it

between if

the

Km of the

two dehydrogenases

flux

does

more

of physiological

and the

accumulation the

fit

of the two dehydrogenases

a variety

activity

as the

impair

of magnitude

are

U/g over

by the normal

such an impairment

two orders

activity

The Km values

substrates

1.5-30

of magnitude

hindered

lactonase

of the two dehydrogenases

respective

the total

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

lactonase unlikely

the two dehydrogenases Km for

the

lactonase

greater.

Acknowledgements. We wish to thank Dr. R. Lagunas for helpful discussion and Miss A. Nieto for valuable technical assistance. P.J.S. was on leave from the School of Biochemistry, University of New South Wales, Kensington, N.S.W., Australia. References 1.

Sapag-Hagar,

M.,

Lagunas,

R. and Sols,

A. (1973)

Biochem.

Biophys.

Res.

Corn. 5&l, 179-185. 2. 3.

Kagawa, 364-371. Kawada,

Y.,

Kagawa,

A. and Shimazono,

N. (1964) J. Biochem.

(Tokyo)

56-,

M., Takiguchi,

H., Kagawa, Y., Suzuki, K. and Shimazono, N. (Tokyo) 51, 405-415. and SmyrniotE, P.Z. (1953) Biochim. Biophys. Acta 12,

(1962) J. Biochem. 4.

Horecker,

B.L.

98-102. 5. 6.

Kawada, Biophys. Glaser,

M., Kagawa, Y., Takiguchi, H. and Shimazono, N. (1962) Biochim. Acta 2, 404-407. L. and Brown, D.H. (1957) J. Biol. Chem. 216, 67-79.

1318