THE EFEECT OF ALLOSTERIC

MODULATORS ON GLYCOLYSIS AND

GLUCONEOGENESIS IN RELATION TO
ENERGY LEVELS IN THE BODY
DR FELICIAN PETER KIBACHA
RESIDENT(PAEDIATRICS AND CHILD
HEALTH)

INTRODUCTION

The pathway of glycolysis can be seen as consisting of 2

separate phases. The first is the chemical priming phase
requiring energy in the form of ATP, and the second is
considered the energy-yielding phase.
In the first phase, 2 equivalents of ATP are used to
convert glucose to fructose 1,6-bisphosphate (F1,6BP).
In the second phase F1,6BP is degraded to pyruvate, with
the production of 4 equivalents of ATP and 2 equivalents
of NADH.

CONT..

The reactions catalyzed by hexokinase, PFK-1 and

PK all proceed with a relatively large free energy
decrease.
These non-equilibrium reactions of glycolysis
would be ideal candidates for regulation of the flux
through glycolysis.
Indeed, in vitro studies have shown all three
enzymes to be allosterically controlled.
In general gllycolysis is inhibited allosterically by
high energy levels ATP/ADP and by the presence
of building blocks in other pathways i.e citrate

HEXOKINASE

Regulation of hexokinase, however, is not the

major control point in glycolysis.

This is due to the fact that large amounts of G6P

are derived from the breakdown of glycogen (the
predominant mechanism of carbohydrate entry
into glycolysis in skeletal muscle) and, therefore,
the hexokinase reaction is not necessary.

Regulation of PK is important for reversing

glycolysis when ATP is high in order to activate
gluconeogenesis. As such this enzyme catalyzed
reaction is not a major control point in glycolysis.
The rate limiting step in glycolysis is the reaction
catalyzed by PFK-1.

PFK1

PFK-1 is a tetrameric enzyme that exist in two

conformational states termed R and T that are in
equilibrium. ATP is both a substrate and an
allosteric inhibitor of PFK-1.

Each subunit has two ATP binding sites, a

substrate site and an inhibitor site. The substrate
site binds ATP equally well when the tetramer is
in either conformation. The inhibitor site binds
ATP essentially only when the enzyme is in the T
state.

F6P is the other substrate for PFK-1 and it also

binds preferentially to the R state enzyme.

CONT..

At high concentrations of ATP, the inhibitor site

becomes occupied and shifting the equilibrium of
PFK-1 conformation to that of the T state
decreasing PFK-1's ability to bind F6P.
The inhibition of PFK-1 by ATP is overcome by
AMP which binds to the R state of the enzyme
and, therefore, stabilizes the conformation of the
enzyme capable of binding F6P.

F2,6BP,PFK2

The most important allosteric regulator of both

glycolysis and gluconeogenesis is fructose 2,6bisphosphate, F2,6BP, which is not an intermediate in
glycolysis or in gluconeogenesis.
The major sites for regulation of glycolysis and
gluconeogenesis are the phosphofructokinase-1 (PFK-1)
and fructose-1,6-bisphosphatase (F-1,6-BPase) catalyzed
reactions.
PFK-2 is the kinase activity and F-2,6-BPase is the
phosphatase activity of the bi-functional regulatory
enzyme,

CONT..

Phosphofructokinase-2/fructose-2,6-bisphosphatase.
PKA is cAMP-dependent protein kinase which
phosphorylates PFK-2/F-2,6-BPase turning on the
phosphatase activity. (+ve) and (-ve) refer to positive and
negative activities, respectively.
The synthesis of F2,6BP is catalyzed by the bifunctional
enzyme phosphofructokinase-2/fructose-2,6bisphosphatase (PFK-2/F-2,6-BPase).
In the nonphosphorylated form the enzyme is known as
PFK-2 and serves to catalyze the synthesis of F2,6BP by
phosphorylating fructose 6-phosphate. The result is that
the activity of PFK-1 is greatly stimulated and the
activity of F-1,6-BPase is greatly inhibited.

CONT..

Under conditions where PFK-2 is active, fructose flow

through the PFK-1/F-1,6-BPase reactions takes place in
the glycolytic direction, with a net production of F1,6BP.
When the bifunctional enzyme is phosphorylated it no
longer exhibits kinase activity, but a new active site
hydrolyzes F2,6BP to F6P and inorganic phosphate.
The metabolic result of the phosphorylation of the
bifunctional enzyme is that allosteric stimulation of PFK1 ceases, allosteric inhibition of F-1,6-BPase is
eliminated, and net flow of fructose through these two
enzymes is gluconeogenic, producing F6P and eventually
glucose.

PKA,cAMP,G PROTEINS

The interconversion of the bifunctional enzyme is

catalyzed by cAMP-dependent protein kinase (PKA),
which in turn is regulated by circulating peptide
hormones.
When blood glucose levels drop, pancreatic insulin
production falls, glucagon secretion is stimulated, and
circulating glucagon is highly increased.
Hormones such as glucagon bind to plasma membrane
receptors on liver cells, activating membrane-localized
adenylate cyclase leading to an increase in the
conversion of ATP to cAMP (see diagram below).
cAMP binds to the regulatory subunits of PKA, leading
to release and activation of the catalytic subunits.

CONT..

PKA phosphorylates numerous enzymes, including the

bifunctional PFK-2/F-2,6-BPase. Under these conditions
the liver stops consuming glucose and becomes
metabolically gluconeogenic, producing glucose to
reestablish normoglycemia.
Representative pathway for the activation of cAMPdependent protein kinase (PKA). In this example
glucagon binds to its' cell-surface receptor, thereby
activating the receptor.
Activation of the receptor is coupled to the activation of
a receptor-coupled G-protein (GTP-binding and
hydrolyzing protein) composed of 3 subunits.

CONT..

The alpha subunit dissociates and binds to and activates

adenylate cyclase. Adenylate cylcase then converts ATP
to cyclic-AMP (cAMP).
The cAMP thus produced then binds to the regulatory
subunits of PKA leading to dissociation of the associated
catalytic subunits. The catalytic subunits are inactive
until dissociated from the regulatory subunits. Once
released the catalytic subunits of PKA phosphorylate
numerous substrate using ATP as the phosphate donor.
Regulation of glycolysis also occurs at the step catalyzed
by pyruvate kinase, (PK). The liver enzyme has been
most studied in vitro. This enzyme is inhibited by ATP
and acetyl-CoA and is activated by F1,6BP

PYRUVATE KINASE
Pyruvate kinase is an enzyme involved in
glycolysis. It catalyzes the transfer of a
phosphate group from phosphoenolpyruvate
(PEP) to ADP,
Yielding one molecule of pyruvate and one
molecule of ATP.

CONT..

The inhibition of PK by ATP is similar to the

effect of ATP on PFK1
The binding of ATP to the inhibitor site reduces
its affinity for PEP.
The liver enzyme is also controlled at the level of
synthesis.
Increases carbohydrate ingestion induces the
synthesis of PK resulting in elevated cellular
levels of the enzyme

CONT..

This step is the final one in the glycolytic

pathway, which produces pyruvate
molecules; the final product of aerobic glycolysis.
However, in anaerobic glycolysis,
lactate dehydrogenase will utilize the NADH
produced by
glyceraldehyde phosphate dehydrogenase to
reduce pyruvate to lactate.
In humans, there are two pyruvate kinase
isozymes: type M (muscle
and type L,R (liver and erythrocyte,
The isozymes differ in primary sequence and
regulation.

CONT..
ATP is a negative allosteric inhibitor.
This accounts for parallel regulation with
PFK 1.

It is not known if citrate plays a role in

negative allosteric inhibition, however it is
believed acetyl-CoA does.

Liver pyruvate kinase is also regulated

indirectly by epinephrine and glucagon,
through protein kinase A. This protein
kinase phosphorylates liver pyruvate kinase
to inactivate it.

GENETIC DEFICIENCY
Genetic defects of this enzyme cause the disease
known as pyruvate kinase deficiency. In this
condition, a lack of pyruvate kinase slows down
the process of glycolysis.

This effect is especially

devastating in cells that lack mitochondria,
because these cells must use anaerobic glycolysis
as their sole source of energy because the
TCA cycle is not available.

cont.
One example is red blood cells, which in a
state of pyruvate kinase deficiency rapidly
become deficient in ATP and can undergo
hemolysis.

Therefore, pyruvate kinase deficiency can

cause hemolytic anemia.

EPINEPHRINE,GLUCAGON AND
INSULIN

Insulin which stimulates uptake of glucose after a

meal stimulates te production of glycolytic
enzymes,involved in the three control points
Glucagon,a pancreatic hormone ,signals a need
for glucose(starvation) and thus inhibits
glycolytic enzymes and stimulates production of
key regulatory enzymes in gluconeogenesis

GLUCONEOGENESIS

CONT...

Gluconeogenesis is the biosynthesis of new glucose, (i.e.

not glucose from glycogen).
The production of glucose from other metabolites is
necessary for use as a fuel source by the brain, testes,
erythrocytes and kidney medulla since glucose is the sole
energy source for these organs.
During starvation, however, the brain can derive energy
from ketone bodies which are converted to acetyl-CoA.
The primary carbon skeletons used for gluconeogenesis
are derived from pyruvate, lactate, glycerol, and the
amino acids alanine and glutamine.
The liver is the major site of gluconeogenesis

CONT..

The three reactions of glycolysis that proceed with a large

negative free energy change are bypassed during
gluconeogenesis by using different enzymes.
These three are the pyruvate kinase, phosphofructokinase1(PFK-1) and hexokinase/glucokinase catalyzed reactions.
In the liver or kidney cortex and in some cases skeletal
muscle, the glucose-6-phosphate (G6P) produced by
gluconeogenesis can be incorporated into glycogen. In this
case the third bypass occurs at the glycogen phosphorylase
catalyzed reaction.
Since skeletal muscle lacks glucose-6-phosphatase it
cannot deliver free glucose to the blood and undergoes
gluconeogenesis exclusively as a mechanism to generate
glucose for storage as glycogen.

Allosteric Modulators in
Gluconeogenesis

Obviously the regulation of gluconeogenesis will be in

direct contrast to the regulation of glycolysis.
In general, negative effectors of glycolysis are positive
effectors of gluconeogenesis.
Regulation of the activity of PFK-1 and F1,6BPase is the
most significant site for controlling the flux toward
glucose oxidation or glucose synthesis.
This is predominantly controlled by fructose-2,6bisphosphate, F2,6BP which is a powerful negative
allosteric effector of F1,6Bpase activity.
Regulation of glycolysis and gluconeogenesis by fructose
2,6-bisphosphate (F2,6BP). The major sites for regulation
of glycolysis and gluconeogenesis are the
phosphofructokinase-1 (PFK-1) and fructose-1,6-

CONT..

PFK-2 is the kinase activity and F-2,6-BPase is the

phosphatase activity of the bi-functional regulatory
enzyme, phosphofructokinase-2/fructose-2,6bisphosphatase.
PKA is cAMP-dependent protein kinase which
phosphorylates PFK-2/F-2,6-BPase turning on the
phosphatase activity. (+ve) and (-ve) refer to positive and
negative activities, respectively.
The level of F2,6BP will decline in hepatocytes in
response to glucagon stimulation as well as stimulation
by catecholamines. Each of these signals is elicited
through activation of cAMP-dependent protein kinase
(PKA)

CONT..

One substrate for PKA is PFK-2, the bifunctional

enzyme responsible for the synthesis and
hydrolysis of F2,6BP.
When PFK-2 is phosphorylated by PKA it acts as
a phosphatase leading to the dephosphorylation of
F2,6BP with a concomitant increase in F1,6Bpase
activity and a decrease in PFK-1 activity.

PK,PC,PEPCK

Secondarily, F1,6Bpase activity is regulated by the

ATP/ADP ratio. When this is high, gluconeogenesis can
proceed maximally.
Gluconeogenesis is also controlled at the level of the
pyruvate to PEP bypass. The hepatic signals elicited by
glucagon or epinephrine lead to phosphorylation and
inactivation of pyruvate kinase (PK) which will allow for
an increase in the flux through gluconeogenesis. PK is
also allosterically inhibited by ATP and alanine. The
former signals adequate energy and the latter that
sufficient substrates for gluconeogenesis are available.
Conversely, a reduction in energy levels as evidenced by
increasing concentrations of ADP lead to inhibition of
both PC and PEPCK. Allosteric activation of PC occurs
through acetyl-CoA.

CONT..

Each of these regulations occurs on a short

time scale, whereas long-term regulation can
be effected at the level of PEPCK.
The amount of this enzyme increases in
response to prolonged glucagon stimulation.
This situation would occur in a starving
individual or someone with an inadequate
diet

BIBLIOGRAPHY

themedicalbiocemistry.org

Stryer principles of biochemistry

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