J. Biol. Chem.-1981-Pilkis-3619-22(1)

8/10/2019 J. Biol. Chem.-1981-Pilkis-3619-22(1)

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3620

Fructose-1 6-bisphosphatasenhibitor

0

IO 2 30

40 5 0

1 2 -

0.9

-

0

5 IO

15

2 0

FRUCTOSE

I,6-BISPHOSPHATE, M MINUTES

FIG.

1 left) .The effect of fru~tose-l,6-[1-~~P]P~oncentra-

tion on the velocity of the

fructose-1,6-bisphosphatase

eac-

tion. Rat liver


as incubated with in-

creasing concentrations of f ru~ tos e-l ,6- [l- ~P ]P~specificactivity, 120

cpm/pmol) for 5 min. The reaction was stopped with acidic ammo-

nium molybdate and the *PI eleased was extracted with butyl acetate

as described under Materials and Methods. The

K ,

for

substrate

was approximately 7

PM.

FIG.

2 center).Time course of fructose-1,6-bisphosphatase-

catalyzed hydrolysis of fru~tose-l,6-[1-~~P]P~n the presence

and absence of fructose-2,6-Pz.Fructose-l,6-bisphosphatase as

incubated with 2.5 p~ fructose 1,6[1-*P]bisphosphate specific activ-

ity, 120 cpm/pmol) in the absence 0 ) nd presence of 1 p~ 0) nd

RESULTS

Effect of Fructose-2,6-P2on the Rat Liver Fructose-l,6-

bisphosphata se Activity-The effect of increasing concentra-

tions of hc to ~e - l , 6 - [ l - ~~ P ] P ~n the rateof release of 32P,y


s shown in Fig.

1.

Under the

conditions of the assay, the substra te oncentration curve was

hyperbolic and half-maximal rates were obtained with about

7

~ M

ructose-1,6-P~. aximum activity was observed with 50

p~ fru~tose-l,6-[1-~~P]P2.

Preliminary experiments using the spectrophotometric as-

say revealed that fructose-2,6-Pzdid not inhibit fructose-l,6-

bisphosphatase activity, but theseexperiments were done

with satu rating concentrations (70 to 150

p ~ )

f fructose-l,6-

Pz.

Fig. 2 shows the time course of 2Pi elease from 2.5

pM

f ru~ tose - l , 6 - [ l -~~P ]P ~atalyzed by fructose-1,6-bisphospha-

tase in the absence and presence of 1

p~

and 5 PM fructose-

2,6-P2. The reaction rates were linear in all cases and both

concentrations of fructose-2,6-P2 inhibited release of

32Pi.

Fructose-2,6-P2 was not a subs trate for fructose-1,6-bisphos-

phatase (3 ,4 ,8 ) , o the inhibition was not due to the presence

of two competing substrates.

Fig.

3

shows the effect of increasing concentrations of fruc-

tose-2,6-Pzon the rateof Pirelease from 2.5pM fructose-1,6-

[1-3P]Ps. Fructose-2,6-Pz was a potent inhibitor

of

fructose-

1,6-bisphosphatase; half-maximal inhibition

of

the enzyme

was observed with about 1 p~ fructose-2,6-P2. Inhibition of

the enzyme was completely prevented by

fwst

incubating

fructose-2,6-P2 at pH 3 for 30 min (Fig. 3 ) . This mild acid

trea tmen t completely hydrolyzes fructose-2,6-P2 o fructose 6-

phosphate and inorganic phosphate 3 , 4, 8). These results

indicate that the inhibition was due to fructose-2,6-P*. This

inhibition was pH-dependent. In the resence of

1p~

fructose-

2,6-P2,25 inhibition was observed at pH 9.2, 65 a t pH 7.5,

and 49 at pH 6.5.

Results of initial ra te studies of fructose-1,6-bisphosphatase

activity in the presence of various concentrations of fructose-

2,6-P2are presented inFig. 4 as double reciprocal (Lineweaver-

Burk) plots of rate uersus fructose l,6-bisphosphate concen-

5 p~ A) ructose-2,6-P~ elease of 32P1 as determined as described

under Materials and Methods.

FIG.

3 right).The effect of acid treatment on the ability of

fructose-2,6-P~ o nhibit

fructose-1,6-bisphosphatase.

ruc-

tose-1,6-bisphosphatase was incubated with 2.5 PM fructose 1,6-[l-

Plbisphosphate (120 cpm/pmol) in the presence of increasing con-

centrations of fructose-2,6-P2

0 )

r

acid-treated fructose-2,6-P~

0).

Th e incubation was terminated after

5

min and the released was

estimated as described under Materials and Methods. Acid treat-

ment of fructose-2,6-Py onsisted of incubating the sugar diphosphate

at pH

3

for 30 min at 22 C . The amount of fructose 6-phosphate

produced had no effect on enzyme activity.

8

64

40

32

16

FIG.4.

Double reciprocal plot of the rate f enzymic hydrol-

ysis of fructo~e-l ,6-[l-~~P]P~emus fructose-1,6-Pzconcentra-

tion in the presence of increasing concentrations

of

fructose

2,6-P2: none

O),0 5 PM

0),1

PM

A), 2.5 PM El), and

5 PM 0

The incubation time was 5 min.

tration. The linear plots showed a common intercept on the

ordinate axis which indicated th at fructose-2,6-P2was a com-

petitive inhibitor of the fructose-1,6-bisphosphataseeaction.

When the resultsof Fig.

4

were analyzed by means of a Dixon

plot, the K , of th is competitive inhibitor was estimated to be

0.5

PM

(data not shown).

Hill plots of the data

from

Fig.

4

are shown in Fig.

5.

The

Hill coefficient, nH,was approximately 1.0 for fructose-1,6-Pz


3/4

Fructose-1,6- bisphosphatase Inhibitor

3621

in the presence or absencef fructose-2,6-P2.This is indicative

of noncooperativity among theites.

Effect of Fructose 2,6-Bisphosphate

on

Inhibition of Fruc-

tose-1,6-bisphosphatase

by

AMP-Although fructose-l,6-bis-

phosphatase exhibits normal Michaelis-Menteninetics with

regard toits substrate (12, 13; Figs.

1

and 4), nhibition of th e

enzyme by AMP has been reported to show

a

high degree of

cooperativity (14, 15) and to be depende nt on the presenc ef

substrat e (16).

It

was of interes t to determin e whether fructose

2,6-bisphosphate could affect the AMP i nhib itio n of the en-

zyme. Fig. 6 illustrates he effect of AMP on fructose-1,6-

bisphosphatase activity with 2.5

,UM fru~tose-l,6-[l-~~P]P.~

s

substrate. AMPby itself did not affect enzyme activity unless

concentrations of grea ter than 0 PM were used. Half-maximal

inhibition was obtained with 200

UM

MP. Previous reports

indicated a Ki for AMP of 10 to 20 ,UM (14-16). Since many of

these studies were done with satura ting fructose ,6-bisphos-

phate concentrations, it was possible that the high K , in the

present study was due o he low substrateconcentration

employed. However, th e presence of 0.5

PM

or 1

PM

fructose

2,6-bisphosphate decreased theK , for inhibition by AMP to

about 18 ,uM. Thus, he presence of low concentrations

of

0 5

I 0

O

5

9

20

8

34

I

7 5

186 2

350

8

I

I

0

I

2

log [ F1.6-PzI

FIG.5. Hill plots for fru~tose-l,6-[l-~~P]P2 in the presencef

increasing concentrations

of

fructose 2.6-bisphosphate.

The

conditions are the same as described in Fig. 4. Beside each plot is the

inhibitor concentration.

>- I

0

40 8 I2

160

200

AMP , p M

FIG.6.

Effect of fructose-2,6-P2 on the nhibition

of

fructose-

1,6-bisphosphatase by AMP.

Fructose hisphosphatase was incu-

bated for 5 min with 2.5 ,UM fructose-1 6-[1-P]P2 and increasing

concentrations of AMP in the absence 0 ) nd presence of 0.5 ,UM

0)

r 1

PM A )

fructose-2 6-P?.jzP, released was determined as

described under Materials and Methods.

fructose-2,6-P2 ncreased the sensitivity of fructose-1,6-bis-

phosphatase to inhibition by AMP. Likewise, a low concen-

tration of AMP (20

p ~

ecreased the concentration of fruc-

tose-2,6-P2 needed for half-maximal inhibition from 0.5

pM

to

0.25

PM

(data not shown). Thisuggests a synergistic interac-

tion between ructose-2,6-Pz and AMP to inhibi tructose- 1,6-

bisphosphatase.

DISCUSSION

The results reported here learly show that fructose-2,6-P2

is a potent competitive inhibitor of

fructose-1,6-bisphospha-

tase. This s perhaps not

so

surprising in view of the resultsof

Benkovic et

al.

(17). They studied the effect of a number

of

structural analogs of fructose-1,6-P2 on fructose-1,6-bisphos-

phatase activity t pH.4. These workers showed ha t replace-

ment of the hydroxyl group at

Cs

by a methoxy group a- nd

/I-methyl-D-fructofuranoside

1,6-bisphosphate) converted the

sugar diphosphate rom a substrate to competitive inhibitor.

Since

fructose-l,-6-bisphosphatase

pecifically hydrolyzes

phosphate from the

C1

position, one would not expect ructose-

2,6-P2 o be asubstrate. However, replacement of t he hydroxyl

group at C, bya phosphate group would be expected to

produce a potent inhibitor. Fr uctose 2,6-P~ has K ,

(0.5 PM

that is one-tenth that f a- nd P-methyl-D-fructofuranoside

1,6-bisphosphate. Th e high affinityof th e enzyme for fructose-

2,6-P2 suggests th at very tight binding of th is effector to the

active site

of

th e enzyme canstill occur. Fructose-1,6-bisphos-

phatase prefers the a-anomerof fructose-1,6-Pz (18)whereas

fructose-2,6-P2 exists as the /I-anomer (8). In both cases, th e

geometry of the phosphatemoieties is in the cisconfiguration

and this could allow both compounds to bind to the enzyme.

Stud ies on the inding of fructose-2,6-P2 to theenzyme in the

presence and absenceof various effectors ar e in progress.

The concentrationof fructose-2,6-Pz required for half-max-

imal activation of phosphofructokinase (3,

4,

8) is similar to

tha t req uir ed for half-maximal inhibition of fructose-1,6-bis-

phosphatase (Figs. 1 and 4) and similar to the concentration

of this effector in isolatedhepatocytes (3,4). Thus, changesn

th e level of fructose-2,6-P2 in hepatocytes would be expected

to affect the activi ty of both enzymes. Glucagon has been

reported to lower th e level of fructose-2,6-P2 in isolated he-

patocytes (3-6) and this may account,

t

least in part, for the

hormone-induced change in substrate cycling that occurs at

this level. Glucagon causesan inhibition of flux through

phosphofructokinase and a stimulation of

flux

through fruc-

tose-l,6-bisphospha tase (19). Th e inhibition of phosphofruc-

tokinase flux has been shown to be due in large par t to the

decrease in ructose-2,6-Pz levels and the resultant nhibition

of phosphofructokinase activity (2-6). I t is possible th at th e

increase in flux through


lso may

be due to the decreasen fructose-2,6-P2 levels. Also, glucagon

addition o hepatocytes has beenshown to enhance phos-

phorylation of both phosphofructokinase (2) and ructose-1,6-

bisphosphatase. The phosphorylation of both enzymes can

be catalyzed by the CAMP-dependent protein kinase in vitro

11,20). In nei ther case has the effect of phosphorylation on

enzyme activity been clearly elucidated. Furuya and Uyeda

(21) have suggested that phosphory lation of phosphofructo-

kinase resul ts in decreased bindingf an activa tor, resumably

fructose-2,6-P2, to th e enzyme. I t is possible tha t th e binding

of fructose-2,6-P2 to both phosphofructokinase and ructose-

1,6-bisphosphatase may e altered by phosphorylation. Work

on these questions isn progress.

T. H. Claus and S . J. Pilkis manuscript in preparation.


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Fructose

1 6-

bisphosphatase Inhibitor

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Documents

J. Biol. Chem.-1981-Pilkis-3619-22(1)