Guanosine-5′-triphosphate as the allosteric effector of fructose 1,6-diphosphatase in Rhodopseudomonaspalustris

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GUANOSINE-5'- TRIPHOSPHATEAS THE ALLOSTERIC EFPECTOROF FRUCTOSE1,6- DIPHOSPHATASEIN RHODOPSEUDOMONAS PALUSTRIS C. S, Stachow and C. F. Springgate Department of Biology, Boston College, Chestnut Hill, Massachusetts, 02167~

Received April 6, 1970 SUMMARY;The inhibition of 300-fold purified fructose 1,6dlphosphatase from Rhodopseudomonas palustris by guanosine5'-triphosphate is described. Kinetic data illustrates that GTP inhibition is specific, allosterlc In nature and noncompetitive. Guanosine-5'-triphosphate was not utilized in the enzymatic reaction. Evidence presented suggests that GTP levels play an important role in regulation of the photosynthetic reductive carbon cycle In Rhodopseudomonas. Adenosine-5'-monophosphate to be an allosteric

(AMP) has been demonstrated

Inhibitor

(Taketa

and Pogell,

1963, 1965;

Underwood and Newsholme, 1965) of fructose

1,6-dlphosphatase

from a wide variety

Mukkada and Bell

(1969)

have reported

Acinetobacter

a fructose

Studies

oycle (Pedersen have provided

et. al., evidence

the light-dark is reported

diphosphatase

1966;

inhlbltion and partially

photoheterotroph

in regulating In this

of fructose

Rsp. palustris. 637

1968, 1969)

diphosphatase biosynthesis

communication 1,6-dlphosphatase

characterized

but AMP-

carbon reduction

Bassham et. al.,

that the fructose

transition.

(FDPase) of

by ATP and citrate

on the photosynthetic

plays a key role

allosterlc

However,

which is inhibited

Insensitive.

reaction

of organisms.

and

the by GTP

in the facultatlve

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MATERIALSANDMEIHODS: Approximately

300-fold

purified

from a.

palustris,

synthetic

medium (Cohen-Bazire

grown photoheterotrophically

and acetate

malate, disrupted operated

ammonium sulfate

buffer,

and streptomycin

column chromatography. will

be published

tion

was stable

free

of the following

Details

for

sulfate

Fructose

enzyme preparaat -150 and was

phosphofructokinase activity

by following

at 340 mu in the presence

buffer

6-phosphate

the rate

(pH 8.0) containing

1.0 ml. All reagents

1.0 mM FDP, 1,O mM

from Sigma Chemical Co, Uniformly purchased

2.pg

and 100 mM Trls-HCL

1.0 mM IXCT in a final

were analytical

from New England Nuclear

of NADP

of excess coupling

contained:

dehydrogenase

and

was deter-

NADP, 1.0 mM MnC12, 10 pg phosphoglucoisomerase, glucose

procedure

enzymes: NADPH oxldase,

1,6-diphosphatase

enzymes. The assay mixture

cell

precipitation,

of the purification

contaminating

mined spectrophotometrically

1.0

and DEAE-cellulose

two weeks when stored phosphatase,

were

by MnC12,

The resulting

elsewhere.

acid and alkaline

reduction

Cells

pH 7.4, containing

FDPase was purified

C-gamma adsorption-elution

aldolase.

1957). Glutamate,

(IYIT) by means of a Wench pressure

at 10,000 psi.

alumina

et, al.,

in

were used as carbon sources,

in 0.1 M Trls-HCL

mM dithlotheitol

FDPase was obtained

volume of

grade and purchased labelled

14C4TP was

Corp.

RESULTS AND DISCUSSION: Inhibition (figure

of fructose

1,6-diphosphatase

1.) was found to be specific

triphosphate

as the following

for guanosine-5'-

compounds tested 638

activtty at a concen-

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Vol. 39, No. 4,197O

Figure

1. Double reciprocal plots of velocity-versus FDP concentration at several GTP concentrations (fixed variable). Curve (A) minus GTPI (B) 1.0 mM GTP; (C) 1.25 mM GTP; (D) 1.5 mM GTP and (?I) 2.0 mM GTP. of 3.0 mM had no effect

tration

3',5'-AHP,

AMP,

cyclic

IMP,

GDP, GMP, cyclic

and NADP,

citrate,

of GTP used enzyme

in

our studies

activity;

CDP, CMP, UTP,

3*,5*-GMP,

plots

reduced

UDP,

ATP, ITP,

ADP,

IDP,

and oxidized

NAD

and pyrldoxal

phosphate.

had no effect

on the

coupling

versus

fructose

1,6-

1.)

several

inhibition

pattern.

does not

influence

8.3 x lo-5

resulted

From this

data

plant

from enzyme

It

Km of FDP which

results

fructose differ

(figure

variable)

the

M. These

of mammalian

1965) but

of velocity

concentration

of GTP (fixed

the

on FDPase

Levels

system,

diphosphate tions

CTP,

acetate

Reciprocal

for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are

similar

1,6-diphosphatase the (Scala,

competitive

at In

a non-competitive

can be seen that was calculated

GTP to be

to the

AMP inhibition

(Taketa

and Pogell,

inhibition

1969). GTP inhibition 639

concentra-

reported could

be

Vol. 39, No. 4,197O

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 1 GTP mM

Figure

on fructose 2, Effect of guanoslne -j'-triphosphate Percent Inhibition of FDPase l,&diphosphatase activity. activity is plotted against varying GTP concentrations at several fixed variable concentrations of FDP. Curve (A) 1.0 mM FDPi (B) 0.4 mM FDP; (C) 0.1 mM FDP.

completely

reversed

concentrations Plots (figure

of percent

Inhibition

s&mold

or approximately series

simple mass action

at varying

the

theory.

The

in 505%inhibition

substrate

levels

of

ranged from

ten times Km. of straight

was plotted

against

coefficient,

indicating further

lines

was

n was calculated of the preoise

binding

obtained

when log

log GTP concentration

more than one GTP binding

The determination await

curves which demonstrate

M at 0.1 mM FDP to 12 x 10J, M at 1.0 mM FDP

7.4 x lOA

The Hill

versus GTP concentration

of GTP which resulted

enzymatic activity

V,-v/v

inhibition

does not follow

concentration

but not by substrate

as high as 20 x Km.

2.) yield

A

by dilution

studies. 640

(figure

3.).

to be 2.9 at 25O site

per enzyme molecule.

number of GTP sites

must

Vol. 39, No. 4,197O

BIOCHEMICAL

-l-

-2

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

/

:

-4

-3 LOG

Figure

-2 bTa

3. Hill plots of GTP Inhibition at various FDP concentrations. Plotted as log Vo-v/v versus log (GTP) where V, represents velocity in the absence of GTP and v represents velocity in the presence of GTP. Curve (A) 0.10 mM FDP; (B) 0.40 mM FDP; (C) 1.0 mM FDP.

To determine 14C labelled

GTP (5.0 mM containing

In the standard Subsequent

whether GTP was utilized reaction

identification

5'-triphosphate Laboratory demonstrated during

mixture

3

x lo6 DPM) was included

and Incubated

and percent

that

15

min.

of guanosine-

(System IV, Pabst

OR-101 and scintillation the Inhibitor,

for

recovery

by paper chromatography Circular

in the reaction,

counting

GTP was not hydrolyzed

the reaction. The regulatory

which is reflected figure

nature

of fructose

in the sigmoid

2, suggests that the levels

play an important

role

reductive

palustris.

to further

Studies

inhibition

mechanism are now in progress

data shown In

of GTP in the cells

in controlling

the photosynthetic

1,6-diphosphatase,

the flow

of carbon in

carbon cycle of Rhodopseudomonas characterize at this

641

this

laboratory.

control

Vol. 39, No. 4,197O

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RKFKRENCES Bassham, J.A., Kirk, M., and Jensen, R.G., Blochim. Biophys. Acta, a, 211 (1968). Bassham, J.A., and Krause, G.H., Biochim. Biophys. Acta, 189, 207 (1969). Cohen-Bazire, G., Slstrom, W.R., and Stander, R.Y., J. Cellular Camp, Physiol., &, 25 (1957). and Bell E.J., Biochem. Biophys. Res. Comm., Muklrada' '&'340 (1969j. Pedersen, T.A., Kirk, M., and Bassham, J.A., Bloohlmi Biophys. Acta, 112, 189 (1966). Scala, J., Ketner, G., and Jyung, W.H., Arch. Biochem, Blophys., Taketa K a, 111 (1969). and Pogell, B.M., Biochem. Biophys. Res. Comm., , '&, 229 (1963). Taketa, K,, and Pogell, B.M., J. Biol. Chem., 240, 651 (1965). Underwood, A.H., and Newsholme, E.A., Biochem. J,, s, 767 Underwood , (,'i"'9.9

and Newsholme, E.A., Biochem.J.,

642

s,

868 (1965).