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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 ratiosATP/ADP(or AMP)NADH/NAD+
NADPH/NADP+
ATP + glucose ADP + glucose 6-phosphate
• AMP is a very sensitive indicator – small changes make a big difference percentage-wise (normal conc. <0.1 mM)
]][[
]6][[ln0
gluATP
PGADPRTGG
-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 points1. 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 6-
phosphate as the other isozymes are
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
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 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 GlycogenPhosphorylase 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” Inactive form “b” Glycogen phosphorylase -P -OHGlycogen synthase -OH -P
Allosteric regulation of GP and GS
GP a (active form) - inhibited by GlucoseGP (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