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8/10/2019 J. Biol. Chem.-1981-Pilkis-3619-22(1)
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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
fructose-1,6-bisphosphatase
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
fructose-1,6-bisphosphatase
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
8/10/2019 J. Biol. Chem.-1981-Pilkis-3619-22(1)
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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
fructose-1,6-bisphosphatase
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.
8/10/2019 J. Biol. Chem.-1981-Pilkis-3619-22(1)
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3622
Fructose
1 6-
bisphosphatase Inhibitor
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