Regulation of Metabolic Pathways
• Systems must respond to conditions
• Homeostasis is not equilibrium
• Dynamic Steady State
– Flux - Rate of metabolic flow of material
through pathways
• Many ways to regulate – for example
– At the protein level (e.g. allosteric control)
– At the gene level
– At transcription or translation
• There are different time scales for regulation
– < sec, seconds, hours, days
– Based on situation that requires response
• Maintaining ATP concentration is critical
– Energy needed to sustain cellular processes
– Typical cell
• [ATP]  5 mM
• ATP-using enzymes KM range 0.1 – 1 mM
• Significant [ATP] drop would cause many
reactions to decrease
• Cells are sensitive to ratios
ATP/ADP(or AMP)
NADH/NAD+
NADPH/NADP+
ATP + glucose  ADP + glucose 6-phosphate
G  G0  RT ln
[ ADP][G6 P]
[ ATP][ glu ]
• AMP is a very sensitive indicator – small changes
make a big difference percentage-wise (normal
conc. <0.1 mM)
-Fast response (sec or less) – usually allosteric
control (faster response than synthesis or
degradation of enzyme)
-Covalent modification (also fast)
most common:
phosphorylation/dephosphorylation
-Slower response (sec to hours) –exterior
effects such as hormones, growth factors
Overall regulatory networks will:
1. maximize efficience of energy source
utilization by preventing futile cycles.
2. partition metabolites between alternative
pathways (Ex: glycolysis and PPP).
3. use the best energy source for the immediate
needs of the cell.
4. shut down biosynthetic pathways when their
products accumulate.
Vocabulary:
Metabolic regulation – maintains homeostasis at
the molecular level (e.g. hold concentrations
of metabolites constant)
Metabolic control – changes flux through a
metabolic pathway
Coordinated Regulation of Glycolysis
& Gluconeogenesis
Futile (substrate) cycles are to be avoided
cycles that recycle metabolites at the expense of
ATP
Glycolysis Regulation
•
•
When ATP is needed, glycolysis is
activated
When ATP levels are sufficient, glycolysis
activity decreases
Control points
1. Hexokinase
2. PFK-1
3. Pyruvate kinase
1.
•
Hexokinase
Hexokinase reaction is metabolically
irreversible
•
G6P (product) levels increase when glycolysis
is inhibited at sites further along in the
pathway
Recall there are 4 isozymes
•
G6P inhibits hexokinase isozymes I, II and III
•
Glucokinase (hexokinase IV) forms G6P in
the liver (for glycogen synthesis) when
glucose is abundant (activity is modulated by
fructose phosphates and a regulatory protein)
• Isozymes I,II and II have similar KM
(important in muscle)
– Normally at saturation
• Hexokinase IV has much higher KM
(important in liver)
– Important when blood glucose is high
• Glucose enters mammalian cells by passive
transport down a concentration gradient from
blood to cells
• GLUT is a family of six passive hexose
transporters
• Glucose uptake into skeletal and heart
muscle and adipocytes by GLUT 4 is
stimulated by insulin
• Other GLUT transporters mediate glucose
transport in and out of cells in the absence of
insulin
• GLUT2 is transporter for hepatocytes
• Quick equilibrium of [glucose] with blood
glucose in both cytosol and nucleus
• Regulator protein – inside the nucleus
– Binds Hexokinase IV and inhibits it
– Protein has regulatory site
• Competition between glucose and
fructose 6-phosphate
– Glucose stimulates release of hexokinase
IV into cytoplasm
– Fructose 6-phosphate inhibits this process
• Hexokinase IV not affected by glucose 6phosphate as the other isozymes are
Addition of a
regulatory
protein raises
apparent KM for
glucose (inhibits
hexokinase IV)
Glucose 6-Phosphate Has a Pivotal Metabolic
Role in Liver
2. Regulation of Phosphofructokinase-1
• Important - this step commits glucose to glycolysis
• PFK-1 has several regulatory sites
• ATP is a substrate and an allosteric inhibitor of
PFK-1 (note that it’s an end-product of the
pathway)
• AMP allosterically activates PFK-1 by relieving the
ATP inhibition (ADP is also an activator in
mammalian systems)
• Changes in AMP and ADP concentrations can
control the flux through PFK-1
•AMP relieves ATP inhibition
of PFK-1
• Elevated levels of citrate (indicate ample
substrates for citric acid cycle) also inhibit
PFK-1
• Most important allosteric regulator is
fructose 2,6-bisphosphate (later in the
chapter)
3. Regulation of Pyruvate Kinase (PK)
• At least 3 PK isozymes exist in vertebrates
• Differ in distribution and modulators
• Inhibited by high ATP, Acetyl-CoA, long-chain
fatty acids (energy in good supply)
Liver form – low blood sugar glucagon 
increased cAMP cAMP-dependent protein
kinase  PK inactivation (is reversed by
protein phosphatase)
• Muscle form – epinephrine→increased cAMP →
activates glycogen breakdown and glycolysis
• PK is allosterically activated by Fructose 1,6 BP
• PK inhibited by accumulation of alanine
Regulation of Gluconeogenesis
• Fate of pyruvate
•Go on to citric acid cycle – requires
conversion to Acetyl Co-A by the pyruvate
dehydrogenase complex
•Gluconeogenesis – first step is
conversion to oxaloacetate by pyruvate
carboxylase
• Acetyl Co-A accumulation
• inhibits
pyruvate dehydrogenase
• activates
pyruvate carboxylase
Coordinated regulation of PFK-1 and FBPase-1
(1) Phosphofructokinase-1 (PFK-1) (glycolysis)
(2) Fructose 1,6-bisphosphatase FBPase-1
(gluconeogenesis)
• Modulating one enzyme in a substrate cycle will
alter the flux through the two opposing pathways
• Two coordinating modulators
•AMP
•Fructose 2,6-bisphosphate
• Inhibiting PFK-1 stimulates gluconeogenesis
• Inhibiting the phosphatase stimulates glycolysis
• AMP concentration coordinates regulation
• stimulates glycolysis
• Inhibits gluconeogenesis
• In the liver, the most important coordinating
modulator is fructose 2,6-bisphophate
(F2,6BP)
• It is formed from F6P by the enzyme
phosphofructokinase-2 (PFK-2)
• It is broken down by the same enzyme, but
at a different catalytic site in the enzyme –
it’s a bifunctional protein
-It is called fructose 2,6-bisphosphatase
(FBPase-2) for this activity
- Balance of PFK-2 to FBPase-2 activity
controlled by
-Glucagon
-Insulin
• F2,6BP stimulates glycolysis
• F2,6BP inhibits gluconeogenesis
Effects of Glucagon and Insulin
The Pasteur Effect
• Under anaerobic conditions the conversion of
glucose to pyruvate is much higher than under
aerobic conditions (yeast cells produce more
ethanol and muscle cells accumulate lactate)
• The Pasteur Effect is the slowing of glycolysis in
the presence of oxygen
• More ATP is produced under aerobic conditions
than under anaerobic conditions, therefore less
glucose is consumed aerobically
Regulation of Glycogen Metabolism
• Muscle glycogen is fuel for muscle contraction
• Liver glycogen is mostly converted to glucose for
bloodstream transport to other tissues
• Both mobilization and synthesis of glycogen are
regulated by hormones and allosterically
• Insulin, glucagon and epinephrine regulate
mammalian glycogen metabolism (hormones)
• Ca2+ and [AMP]/[ATP] (muscle glycogen
phosphorylase)
• [glucose] (liver glycogen phosphorylase)
• [glucose 6-phosphate] (glycogen synthase)
• Hormones
•Insulin is produced by -cells of the pancreas
(high levels are associated with the fed state)
-increases glucose transport into muscle,
adipose tissue via GLUT 4 transporter
-stimulates glycogen synthesis in the liver
• Glucagon is Secreted by the a cells of the
pancreas in response to low blood glucose
(elevated glucagon is associated with the
fasted state)
-Stimulates glycogen degradation to
restore blood glucose to steady-state
levels
-Only liver cells are rich in glucagon
receptors
•
Epinephrine (adrenaline) Released from the
adrenal glands in response to sudden energy
requirement (“fight or flight”)
- Stimulates the breakdown of glycogen to
G1P (which is converted to G6P)
-Increased G6P levels increase both the rate
of glycolysis in muscle and glucose
release to the bloodstream from the liver
Reciprocal Regulation of Glycogen
Phosphorylase and Glycogen Synthase
• Glycogen phosphorylase (GP) and glycogen
synthase (GS) control glycogen metabolism in liver
and muscle cells
• GP and GS are reciprocally regulated both
covalently and allosterically (when one is active the
other is inactive)
• Covalent regulation by phosphorylation (-P) and
dephosphorylation (-OH)
COVALENT MODIFICATION (Hormonal control)
Active form “a”
Glycogen phosphorylase
Glycogen synthase
Inactive form “b”
-P
-OH
-OH
-P
Allosteric regulation of GP and GS
GP a (active form) - inhibited by Glucose
GP (muscle)- stimulated by Ca2+ and high [AMP]
GS b (inactive form) - activated by Glucose 6-Phosphate
• Hormones initiate enzyme cascades
•Catalyst activates a catalyst activates a
catalyst, etc.
• When blood glucose is low: epinephrine and
glucagon activate protein kinase A
• Glycogenolysis is increased (more blood glucose)
• Glycogen synthesis is decreased