Control of Metabolism Page 1 of 12

Metabolic Control and Integration of Metabolism

Introduction
There are three reasons why control is necessary in metabolic pathways:
1. To avoid substrate (“futile”) cycles.
For example, the constant interconversion of fructose 6-phosphate
to fructose 1,6-bisphosphate and vice versa, which consumes ATP.

The “useless flow” of metabolites through such cycles is useful in

that it allows rapid increases in flux through a given pathway. By
slightly altering the rate of either of the two enzymes, huge
variations in the net flux can be obtained, if there was originally a
large futile flux through both the enzymes. [For example, the flux
down the glycolytic pathway has been suggested to increase as
much as 1000-fold at the initiation of intense exercise. Allosteric
activation of enzymes alone seems unlikely to explain this increased
flux!] These cycles can also be useful in generating heat from the
hydrolysis of ATP. For example, bumblebees must keep a thoracic
temperature of about 30° C to be able to fly. It turns out that their
bisphosphatase is not inhibited by AMP, which seems to suggest it
evolved for heat production.
2. To link energy production to energy usage.
3. To respond to physiological changes

In controlling catabolic vs. metabolic pathways, effective control can only

happen at irreversible steps. At these steps, the forwards and backwards
reactions are catalysed by distinct enzymes, which means that the activity of
one can be increased while that of the other is decreased. In totally reversible
steps, changing the activity of the enzyme changes the rate of the forwards
and backwards steps equally.

Enzyme activity is controlled in two ways:

© Daniel Guetta 2007, some diagrams from Stryer

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 The amount of enzyme can be changed by tweaking its rate of

synthesis or rate of destruction.

In mammals, however, this is a fairly slow process, and t½ can range

from hours to days. Thus, it tends to occur as a result of long term
changes. For example:
o An increase in the lipoprotein lipase in lactating mammary glands.
o Changes in liver enzymes during the shift from the fed state to
starvation.
o Increases in drug-metabolising enzymes following the intake of
foreign compounds.

In bacteria, however, induction of enzyme can be rapid. For example,

when E. coli is exposed to lactose, the induction of β-galactosidase
occurs rapidly (genes can quickly be activated and deactivated).
 Metabolic control of the enzyme is a much more rapid response. It is
often the case that the product of a pathway inhibits the committed
step (the first non-reversible step) of the pathway. This prevents
intermediates accumulating, and preserves the reagent of the cycle.

Enzyme reaction rates can be controlled in two ways:

o Allosteric regulation is particularly important in metabolic
regulation. The binding of an allosteric effector changes the
affinity of the enzyme of its substrate(s). The effect can be
positive (increase in affinity) or negative (decrease in affinity),
and is very fast.

© Daniel Guetta 2007, some diagrams from Stryer

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o Covalent modification most commonly involves phosphorylation

by protein kinases. This causes a conformational change in the
protein. [Less important in prokaryotes]. One of the main reasons
this is an important form of enzyme control is because it is
usually the final step in a cascade of reactions, that are triggered
by a very low concentration of a signal molecule.

This provides two levels of control within the cell:

o Allosteric regulation is very rapid, and signals are usually
intracellular.
o Phosphorylation is usually regulated by extracellular agents (eg:
hormones). For example, glucagon, insulin and adrenaline are
particularly important in controlling fat and carbohydrate
metabolism. Glucagon and insulin are produced in response to low
and high glucose levels, and adrenaline is released from the
adrenal gland and stimulates the release of food reserves.

Other ways to control metabolic pathways (not in lecture notes) are:

 Compartmentation.
 Specialisation of organs.

Metabolic Control in Glycolysis

The control of glycolysis will vary according to the type of tissue:
 Muscle uses glycolysis to generate ATP.
 The liver produces nearly no energy, but it is a net producer of glucose
in the fasted state, and synthesises triglycerides and glycogen in the
fed state.

The metabolic control can be considered in key stages.

© Daniel Guetta 2007, some diagrams from Stryer

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Transport of Glucose into the Cell

Different transporters mediate the thermodynamically downhill
movement of glucose across the plasma membrane of animal cells.
Different members of the family have distinctive roles:
 GLUT1 and GLUT3 are present in nearly all mammalian cells,
and are responsible for basal glucose uptake. Their KM value for
glucose is 1 mM, significantly less than the normal serum-glucose
level. Hence, these transport glucose into cells are a roughly
constant rate.
 GLUT2 is present in liver and pancreatic β cells is insulin
independent but has a very high KM value (about 15-20 mM).
Thus, glucose enters these tissues only when there is much
glucose in the blood. Thus, the pancreas can sense the glucose
level, and liver cells only utilise glucose to produce triglycerides
and glycogen when it is abundant.
 GLUT4, which has a KM of 5 mM transports glucose into muscle
and fat (adipocyte) cells. However, in the absence of insulin, these
transporters are trapped inside intracellular vesicles. Insulin
recruits these vesicles to the cell membrane, allowing the
transport of glucose.
 [GLUT5, in the small intestine, functions primarily as a fructose
transporter].

Control at Phosphofructokinase
This is the most important control site in the mammalian glycolytic
pathway. The reason for this is that it is the committed step in
glycolysis, despite the fact that it is not the first reaction. The reaction
catalysed by hexokinase does not have this status simply because glucose
6-phosphate is not only a glycolytic intermediate – it can also be
converted to glycogen and used in the OPPP.
 This enzyme is inhibited as the energy charge (ATP/AMP
ratio) rises:

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o ATP is both a substrate and an allosteric inhibitor of

phosphofructokinase-1. ATP binds to a specific regulatory
site that is distinct from the catalytic site.
o AMP, however, reverses the inhibitory action.
This is perfect, because in muscle cells, ATP is primarily needed
to power contraction, a process which requires a high energy
charge.

[Note that it is AMP and not ADP which is the positive regulator
of the enzyme. This is first of all because adenylate kinase
catalyses the following reaction in muscles, to salvage some ATP
back:


ADP + ADP  ATP + AMP
Furthermore, the use of AMP provides very sensitive control.
This is because the concentration of the adenylate pool stays
roughly constant over time. The concentration of ADP, however,
is much higher than the concentration of AMP. Thus, a small
percentage change in [ATP] results in a much larger percentage
change in [AMP] than it does in [ADP].]
 Low pH also enhances the inhibitory effect of ATP in muscular
phosphofructokinase. Such acidic environments could be produced
by high concentrations of lactate. In such conditions, muscle must
halt glycolysis to prevent damage to muscle tissue. In the liver,
lactate is rarely formed.
 Citrate enhances the inhibitory effect of ATP in liver
phosphofructokinase. This is, again, ideal, because high [citrate]
signifies that the citric acid cycle is “filling up”, and that no
further glycolytic products are required for biosynthesis.

Control at Hexokinase
Hexokinase has a very low KM indeed (about 0.1 mM) and so can
operate well under low glucose conditions. However, it is inhibited by its
product, glucose-6-phosphate. It signals that the cell no longer needs

© Daniel Guetta 2007, some diagrams from Stryer

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G6P for biosynthesis or energy. G6P is the way phosphofructokinase

communicates with hexokinase.

The liver, however, also possesses an isozyme of hexokinase, called

glucokinase, which has two key differences:
 It is not inhibited by glucose-6-phosphate. Thus, it can provide
glucose-6-phosphate for the synthesis of glycogen and fatty acids,
two of the major roles of the liver, even if no energy is needed.
 The KM value of glucokinase is very high (10 mM). This ensures
that the brain and muscle have first call on glucose when it is
sparse. [The liver, for example, will not re-absorb the low
concentrations of glucose that it releases during fasting!]

Control at Pyruvate Kinase

This enzyme catalyses the last irreversible reaction of glycolysis.
 ATP allosterically inhibits the reaction to slow it when energy
charge is high.
 Alanine (synthesised in one step from pyruvate) also inhibits
pyruvate kinase. In this case, to signal that building blocks are
abundant.
 Fructose 1,6-bisphosphate (the product of the previous
irreversibly reaction) activates this enzyme, to make sure it is
able to keep pace with the oncoming high flux of intermediates.
This is an example of “feedforward stimulation”.
 In the liver, the L (as opposed to the M) isoenzyme is present,
which can be phosphorylated and therefore made less active. This
occurs as a result of the cyclic AMP cascade triggered by
glucagon (which is released when blood glucose levels are low).

Glycolysis and Gluconeogenesis

Glycolysis and gluconeogenesis are reciprocally regulated. Energy charge will
determine which is most active at two points (these, of course, are the

© Daniel Guetta 2007, some diagrams from Stryer

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irreversible points in the pathways, which therefore have substrate cycles

associated with them.

Reciprocal control at Pyruvate Kinase

We mentioned above that pyruvate kinase is activated by F-1,6-BP and
alanine. It turns out that pyruvate carboxylase, one of the enzymes
catalysing the opposite reaction, is inhibited by ADP and activated by
Acetyl CoA.

The presence of cAMP (as a result of glucagon or adrenaline) also causes

this enzyme to be phosphorylated, which inhibits its activity.

Reciprocal control at Phosphofructokinase

The enzyme that catalyses the opposite reaction is fructose-1,6-
bisphosphatase. As expected, it turns out that this enzyme is inhibited
by AMP and activated by citrate.

There is another factor, however, which we have not yet taken into
account; that is the regulation through fructose-2,6-bisphosphate. This
molecule is synthesised from fructose 6-phosphate:
phosphofructokinase 2 (PFK2)

Fructose 6 - phosphate 
fructose bisphosphatase 2 (FBPase2)

 Fructose 2,6 - bisphosphate

This molecule activates phosphofructokinase (and therefore glycolysis)

and inhibits fructose 1,6-bisphosphate (and therefore gluconeogenesis).

Several factors control the synthesis and breakdown of this molecule:

 Large quantities of fructose 6-phosphate increase the rate of
production of fructose 2,6-bisphosphate (since fructose 6-
phosphate is the substrate of the synthesis, AND since it
stimulates phosphoprotein phosphatase which de-phosphorylates
the double-headed enzyme – see below) This is another example of
feedforward stimulation.

© Daniel Guetta 2007, some diagrams from Stryer

 Control of Metabolism Page 8 of 12

 The blood glucose level has an effect on the production of fructose

2,6-bisphosphate in the liver. Low blood glucose level causes the
release of the hormone glucagon into the blood, which results in a
cAMP cascade. This activates FBPase2 and inhibits PFK2,
decreases the concentration of fructose 2,6-bisphosphate in the
liver and increases the rate of gluconeogenesis. The same occurs
vice-versa. [Note that adrenaline has a similar effect on the liver].

Interestingly, both these enzymes are on a single polypeptide

chain! Which part of the chain is active at one point depends on
the phosphorylation of a single serine residue. When the residue is
phsophorylated, FBPase2 is activated and PFK2 is inhibited, and
vice versa. The addition and removal of the phosphate group is
catalysed by protein kinase A (addition) and phosphoprotein
phosphatase (removal).

Note also that a substrate cycle exists at this point, and so small
changes in both the enzymes caused by one of the factors described
above can cause large changes in the flux. In muscle, this cycle is
controlled by AMP.

Glycogen Synthesis
A few points worthy of attention:
 Glycogen synthase is activated by insulin and inhibited by cAMP (as a
result of glucagon or adrenaline).
 Glycogen phosphorylase is activated by cAMP and AMP, but inhibited
by ATP.

The Citric Acid Cycle

The citric acid cycle is central in many ways, and is controlled at several
points:

© Daniel Guetta 2007, some diagrams from Stryer

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Respiratory Control
First, the tight coupling of the citric acid cycle and the electron
transport chain is an important control mechanism (see TCA cycle
notes).

Pyruvate Dehydrogenase
Pyruvate dehydrogenase is the point of no return for glucose derived
carbon, because Acetyl CoA cannot be turned back to glucose. This
conversion essential commits the carbon to two principle fates –
oxidation to CO2 and incorporation into lipid.

This critical enzyme is stringently controlled:

 Acetyl CoA inhibits the transacetylase (E2) compoenent of the
enzyme by direction binding.
 NADH inhibits the dihydropropyl dehydrogenase (E3)
component.
These two conditions inform the cell that the energy needs of the cell
have been met, or that the cell is degrading fatty acids to produce
NADH and Acetyl CoA. In this case, glucose can be spared. [Note that
it is the energy charge that is important here].

The key means of regulation, however, is covalent modification. PDH

kinase and PDH phosphatase catalysed the phosphorylation and
dephosphorylation respectively of the pyruvaste dehydrogenase (E1)
compoenent of the complex. These are associated to the complex.
 The kinase is inhibited by pyruvate, CoA, NAD+, but activated
by acetyl CoA, NADH and ATP (the immediate and eventual
products of this enzyme’s action).
 The phosphatase is activated by Ca2+ (the signal that gives rise to
muscle contraction). Similarly, insulin (= fed state) stimulates
this enzyme in tissues capable of fat synthesis (eg: adipocytes),
since they synthesise lipids from Acetyl CoA.

© Daniel Guetta 2007, some diagrams from Stryer

 Control of Metabolism Page 10 of 12

Citrate Synthase
This enzyme is allosterically inhibited by ATP (ATP increases KM of the
enzyme for Acetyl CoA). This is important for gluconeogenesis during
starvation; if the energy charge is high, then oxaloacetate can be used
for gluconeogenesis, and Acetyl CoA can be used to generate ketone
bodies. Both of these pathways can be used to fuel the brain (the latter
only in an emergency).

Isocitrate Dehydrogenase
This is inhibited by ATP and NADH (which displaces NAD+), and
activated by ADP and NAD+. [A great illustration of control – if this is
inhibited, citrate then builds up and travels to the cytoplasm where it
inhibits glycolysis and can act as a source of Acetyl CoA for fatty acid
synthesis.]

a-ketoglutarate dehydrogenase
This is inhibited by high energy charge and by its products – succinyl-
CoA and NADH, but activated by Ca2+.

Metabolic Control Analysis

Often, the analysis of metabolic enzymes separately cannot generate a
complete picture of how the overall system is controlled. For example,
increasing the amount of enzyme for ethanol production in yeast do not
increase the amount of ethanol produced, but increasing the need for
ATP does.

Metabolic control analysis attempts to predict the effect of varying one or

more of the enzymes of a pathway. The concept of demand control is often
used.

© Daniel Guetta 2007, some diagrams from Stryer

 Control of Metabolism Page 11 of 12

For example, if flow is through three enzymes, the first and that last of which
catalyse reactions near equilibrium whereas the middle one catalyses the
forwards reaction very efficiently, then it is the concentration of the middle
enzyme that will have the potential to control flux in the pathway, even if
flux through that enzyme is much less. In other words, it has the largest flux
control coefficient, which is defined as the rate of fractional changes in flux
with respect to the fractional changes in enzyme concentration (ranges from 0
– no effect on the flux, to 1 – all the effect on the flux):
¶J / J
¶[E ]/[E ]
The sum of all the flux control coefficients involved in controlling a pathway
must be equal to 1. This is the additivity theorem of metabolic control.

For glycolysis, it turns out that even though PFK-1 is a “key control enzyme”,
the rate of lactate production is not simply controlled by this enzyme because
of the influence of others. Feedback inhibition may operate, and metabolites
may flow into other pathways. MCA demonstrates that a large number of
parameters affect the rate of lactate production and there is no rate
determining step!

When acetate is the only carbons source of the cell, citrate synthase has a flux
control coefficient close to 1. It seems to have less of an impact when glucose
is the energy source.

Three major intermediates

Glucose 6-phosphate
As soon as glucose enters into the cell, it is converted to glucose 6-
phosphate. This prevents its exit across the cell membrane. Note that
that entry of glucose 6-phosphate into the glycolytic pathway can be
both catabolic (when this is done for energy) or anabolic (when this is
done to provide carbon skeletons).

© Daniel Guetta 2007, some diagrams from Stryer

Control of Metabolism Page 12 of 12

Glycogen

Glucose 1-phosphate

Glucose Glucose 6-phosphate OPPP

Fructose 6-phosphate Ribose 5-

phosphate

Pyruvate

Pyruvate and Acetyl CoA

A few notes:
 The conversion to lacatate regenerates redox balance and
basically buys time, and shifts the metabolic burden to other
organs.
 The conversion of pyruvate to alanine is a transamination, and
several other amino acids can be transaminated to pyruvate. Thus,
transamination is a major link between amino acid and
carbohydrate metabolism.

© Daniel Guetta 2007, some diagrams from Stryer

 Will vary depending on type of cell being considered
Intro Muscle uses glycolysis to produce energy
Liver - net producer of glucose, triacylglycerides and glycogen
Glycolysis and gluconeogenesis reciprocally regulated at substrate cycles
Family of glucose transporters
In all cells
Insulin independent
GLUT1 and GLUT3 Km 1 mM (< normal serum
glucose level)
Basal uptake

Present in liver and pancreas

Transport of
glucose into cell Insulin independent
Km 15-20 mM
GLUT2
Will only take up glucose if it's in high concentrations
Allows liver to synthesise fats only when
needed, and pancreas to detect glucose

Km of 5 mM
GLUT4 Transports glucose into fat and muscle cells
However, trapped inside cell in vesicles unless insulin present

Most important because it's the first

Previous steps can also go to OPPP
committed step of glycolysis

e.g.: increasing amount of enzyme for ethanol ATP is an allosteric inhibitor

production in yeast doesn't increase ethanol produced (separate regulatory site)
Metabolic control analysis predicts the effect on Inhibited by high energy charge
AMP relieves this inhibition
flux of varying one enzyme in a pathway
If A and C catalyse reactions close to ADP + ADP --> ATP + AMP
equilibrium (rate fd = rate bk) but B
Very sensitive control
catalyses a forward reaction For example, if
AMP used rather than ADP as inhibitor Less AMP than ADP in cell
Changing flux slightly through B will have more reaction A --> B --> C
of an effect than changing slightly through A or C Often, analysing enzymes Therefore, small change in [AMP]
individually isn't good enough is a huge precentage change
Metabolic control analysis
In muscle, low pH also enhances If too much lactate is being produced
Flux control coeffcient is inhibitory capacity of ATP
"rate in fractional change of Don't want to damage the muscle
flux" with respect to " fractional
For a given substrate
change in enzyme conc " TCA is "filling up"
Sum of all coefficients must be 1 - In liver, high citrate also inhibits it
additivity theorem of metabolic control
For glycolysis, for example, there is NO rate determining step Inhibited by ADP
Fructose 1,6 bisphosphatase
Phosphofructokinase Activated by citrate

Glycolysis Activates glycolysis and inhibits gluconeogenesis

Tight control between electron transport chain and TCA is important through the enzymes at this point
Respiratory control
Made by phosphofructokinase 2 (PFK 2)
Point of no return Destroyed by fructose bisphosphatase 2 (FBPase 2)
Commits it to either oxidation to CO2 or incorporation in lipid Control of Which is active depends on
Stringently controlled Metabolism phosphorylation of single serine residue
Both enzymes on one
Acetyl CoA inhibits E2 (transacetylase) function If "P", FBPase 2 active, and PFK 2 not
polypeptide chain
NADH inhibits E3 (dihydrolipoyl dehydrogenase) function Catalysed by protein kinase A and
Control through fructose
phosphoprotein phosphatase
Most important control mechanism 2-6 bisphosphate
Pyruvate dehydrogenase
PDH kinase Increases production
(deactivates of F 2, 6 BP
Activated by NAD+, CoA and pyruvate
enzyme) F6P is the substrate for PFK2
Inhibited by NADH, ATP, Acetyl CoA Covalent modification of E1
Fructose 6-phosphate
TCA Also activates phosphoprotein phosphatase
Activated by Ca2+ (muscle
contraction) and insulin Feedforward stimulation
PDH phosphatase
Low glucose --> glucagon --> cAMP --> activates FBPase 2
Blood glucose level
Alosterically inhibited by ATP (increases Km of enzyme for Acetyl CoA)
Citrate synthase
Important in starvation - can use oxaloacetate for gluconeogenesis Very low Km - can operate in
low glucose concs
Inhibited by ATP and NADH (which displaces AND+ in the active site)
Not needed for biosynthesis or energy
Muscle
Activated by ADP Inhibited by G6P
Isocitrate dehydrogenase This is the way phosphofructokinase
Great illustration of control - citrate builds up and travels to communicates with hexokinase
cytoplasm, where it halts glycolysis Hexokinase
Very high Km (~ 10 mM) ensure that the
Inhibited by high energy charge and Succinyl CoA and NADH ALPHA-ketoglutarate brain and muscle get first call on glucose
dehydrogenase
Activated by Ca2+ G6P constantly made for synthesis
Liver
of glycogen and fatty acids
Not inhibited by G6P
Even if not *needed* for energy

ATP - no need for more energy

Inhibited by Alanine - made in single step
from pyruvate

Fructose 1,6 bisphosphate

Activated by Product of last irreversible reaction
Feedforwards stimulation
Pyruvate kinase Can be phosphorylated and inactivated
In liver, L (instead of e.g.: as a result of production of
M) isozyme present glucagon which produces a
cAMP cascade
Glucose is low!

Inhibited by ADP
Pyruvate carboxylase
Activated by Acetyl CoA

Subtopic
Glycogen synthesis

(C) Daniel Guetta, 2006