Transcript Glycogen Metabolism and Gluconeogenesis

Glycogen Metabolism and
Gluconeogenesis
CH 339K
Glycolysis (recap)
• We discussed the reactions which convert glucose
to pyruvate:
C6H12O6 +2 NAD+ + 2 ADP  2 CH3COCOOH + 2 NADH +2 ATP + 2 H+
• What about the sources of glucose?
– Dietary sugars
– Glycogen
Before we get to glycogen: Dietary sugars
Starches
Pancreatic Amylase
Maltose
Salivary Amylase
Glucose
Maltase
Maltose
Glucose
Glucose Isomerase
Sucrose
Sucrase
Fructose
Lactase
Lactose
Glucose
Glucose Epimerase
Galactose
Amylase Reaction
Glycogen
• Branched every 8-12
residues
• Up to 50,000 or so
residues total
Breakdown: Glycogen Phosphorylase
Glycogen Synthesis and Breakdown
• Glycogen synthesis and breakdown are both
controlled by hormones
• Glucagon, Epinephrine
– turn on glycogen breakdown
– Turn off glycogen synthesis
• Hormones act through receptors on cell
surface and G-proteins
Glucagon – 29 amino acid polypeptide
produced in pancreas in response to
low blood sugar
Epinephrine – aka adrenaline –
produced by adrenal medulla in
response to stress
Activation of Glycogen Phosphorylase
• G-Proteins
• Second messengers
• Kinase Cascade
3’-5’ cyclic AMP
G-Proteins
G proteins are heterotrimers,
containing Ga, Gb and Gg
subunits.
Subunit
Size
Ga
45 – 47 kD
Gb
35 kD
Gg
7-9 kD
G-Proteins
•
The Ga subunits bind guanine nucleotides (hence the name “G Protein”). G
Proteins are associated on one hand with the inner surface of the plasma
membrane, and on the other hand with membrane spanning receptor proteins
called G-protein coupled receptors or GPCRs.
•
There are a number of different GPCRs; most commonly these are receptors for
hormones or for some type of extracellular signal.
•
In the “resting” state, Ga is bound to the Gb-Gg dimer. Ga contains the
nucleotide binding site, holding GDP in the inactive form, and is the “warhead” of
the G protein. At least 20 different forms of Ga exist in mammalian cells.
•
Binding of the extracellular signal by the GPCR causes it to undergo an
intracellular conformational change; this causes an allosteric effect on Ga. The
change in Ga causes it to exchange GDP for GTP. GTP activates Ga, causing
it to dissociate from the Gb-Gg dimer. The activated Ga binds and activates an
effector molecule.
•
Ga also has a slow GTPase activity. Hydrolysis of GTP deactivates Ga, which
reassociates with the Gb-Gg dimer and the GPCR to reform the resting state. In
other words, G-protein mediated cellular responses have a built-in off switch to
prevent them from running forever.
G-Protein Coupled Receptors (GPCRs)
G-Proteins – Effect of GDP/GTP Binding
GDP – missing terminal
PO4 allows the bg-binding
loop (red) to assime a
looser conformation
GTP – terminal PO4
constrains the bg-binding
loop (red)
Cycling of G protein between active
and inactive states
G-Protein Killers
Cholera
Cholera toxin secreted by the bacterium Vibrio cholera.
A subunit and five B subunits.
A subunit catalyzes the transfer of an ADP-ribose from NAD+ to a specific Arg side
chain of the α subunit of Gs.
Ga is irreversibly modified by addition of ADP-ribosyl group;
Modified Gα can bind GTP but cannot hydrolyze it ).
As a result, there is an excessive, nonregulated rise in the intracellular cAMP level
(100 fold or more), which causes a large efflux of Na+ and water into the gut.
Pertussis (whooping cough)
Pertussis toxin (secreted by Bordetella pertussis) catalyzes ADP-ribosylation of a
specific cysteine side chain on the α subunit of a G protein which inhibits adenyl
cyclase and activates sodium channels.
This covalent modification prevents the subunit from interacting with receptors; as a
result, locking Gα in the GDP bound form.
You probably vaccinate your dog against the related species that causes kennel
cough.
Cholera is still a problem2009 Zimbabwe outbreak – 4300 deaths
Activation of Adnylate Cyclase
Activation of cAMP-Dependant Protein Kinase
Glycogen Phosphorylase
• Exists in 2 forms
– Phosphorylase B (inactive)
– Phosphorylase A (active)
• Phosphorylase B is converted to Phosphorylase
A when it is itself phosphorylated by Synthase
Phosphorylase Kinase (SPK)
• GP cannot remove branch points (a-1,6
linkages)
Activation of Glycogen Phosphorylase
cAMP – dependent
Protein Kinase
3’-5’ cyclic AMP
Activation of Glycogen Phosphorylase
PLP: Pyridoxal
Phosphate cofactor
cAMP – dependent
Protein Kinase
Debranching Enzyme
•
•
•
•
The activity of phosphorylase ceases 4 glucose residues from the
branch point.
Debranching enzyme (also called glucan transferase) contains 2
activities:
– glucotransferase
– glucosidase.
Glycogenolysis occurring in skeletal muscle could generate free
glucose which could enter the blood stream.
However, the activity of hexokinase in muscle is so high that any free
glucose is immediately phosphorylated and enters the glycolytic
pathway.
Cori Disease
• Cori disease (Glycogen storage disease Type
III) is characterized by accumulation of
glycogen with very short outer branches,
caused by a flaw in debranching enzyme.
• Deficiency in glycogen debranching activity
causes hepatomegaly, ketotic hypoglycemia,
hyperlipidemia, variable skeletal myopathy,
cardiomyopathy and results in short stature.
Glycogen Synthesis
• Glycogen Synthase adds glucose residues to
glycogen
• Synthase cannot start from scratch – needs a primer
• Glycogenin starts a new glycogen chain, bound to
itself
Glycogen Synthesis (cont.)
• Synthase then adds to the nonreducing end.
Glycogen Synthesis (cont.)
• To add to the glycogen
chain, synthase uses an
activated glucose, UDPGlucose
• UDP-Glucose
Pyrophosphorylase
links UDP to glucose
Glycogen Synthesis (cont.)
• Synthase then adds the activated glucose to the
growing chain
• Release and subsequent hydrolysis of
pyrophosphate drives the reaction to the right
Glycogen Synthesis (cont.)
• Glycogen branching enzyme then introduces
branch points
Mature Glycogen
• Built around
glycogenin core
• Multiple nonreducing ends
accessible to
glycogen
phosphorylase
Reverse Regulation of Phosphorylase
and Synthase
• The same kinase
phosphorylates both
glycogen phosphorylase
and synthase
• Synthase I (dephos.) is
always active
• Synthase D (phos.) is
dependent on [G-6-P]
• The same event that
turns one on turns the
other one off.
Gluconeogenesis
CH 339K
Gluconeogenesis
• Average adult human uses 120 g/day of
glucose, mostly in the brain (75%)
– About 20g glucose in body fluids
– About 190 g stored as glycogen
– Less than 2 days worth
• In addition to eating glucose, we also make it
• Mainly occurs in liver (90%) and kidneys
(10%)
• Not the reverse of glycolysis
• Differs at the irreversible steps in glycolysis
Gluconeogenesis
Differs Here
And Here
And Here
First
Difference
Glycolysis: make a
nucleotide
triphosphate
Gluconeogenesis:
burn two nucleotide
triphosphates
Pyruvate Carboxylase
PEP Carboxykinase
Malate Shuttle
• Pyruvate Carboxylase
is mitochondrial
• OAA reduced to malate
in matrix
• Carrier transports
malate to cytoplasm
• Cytoplasmic malate
dehydrogenase
reoxidizes to OAA
• Mammals have a
mitochondrial PEPCK
Second and Third differences
Energetics
Gluconeogenesis
•
Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H2O ⇌ glucose + 4 ADP + 2 GDP + 2 NAD+
• G = -37 kJ/mol
Glycolysis (reversed)
•
Pyruvate + 2 ATP + 2 NADH + 2 H2O ⇌ glucose + 2 ADP + 2 NAD+
• G = +84 kJ/mol
Net difference of 4 nucleotide triphosphate bonds at ~31 kJ
each accounts for difference in Gs
Local Regulation
• Phosphofructokinase-1(Glycolysis) is
inhibited by ATP and Citrate and stimulated
by AMP.
• Fructose-1,6-bisphosphatase
(Gluconeogenesis) is inhibited by AMP.
Global Control
Enzymes relevant to these pathways that are
phosphorylated by cAMP-Dependent Protein
Kinase include:
• Pyruvate Kinase, a glycolysis enzyme that is
inhibited when phosphorylated.
• A bi-functional enzyme that makes and
degrades an allosteric regulator, fructose2,6-bisphosphate.
Pyruvate Kinase Regulation
• Local regulation by substrate activation
• Global regulation by hormonal control of Protein
Kinase A
Effects of Fructose-2,6-Bisphosphate
• Fructose-2,6-bisphosphate allosterically activates the glycolysis
enzyme Phosphofructokinase-1, promoting the relaxed state,
even at relatively high [ATP]. Activity in the presence of fructose2,6-bisphosphate is similar to that observed when [ATP] is low.
Thus control by fructose-2,6-bisphosphate, whose concentration
fluctuates in response to external hormonal signals, supercedes
control by local conditions (ATP concentration).
• Fructose-2,6-bisphosphate instead inhibits the
gluconeogenesis enzyme Fructose-1,6-bisphosphatase.
Source of Fructose-2,6-Bisphosphate
Fructose-2,6-bisphosphate is synthesized and degraded by a bifunctional enzyme that includes two catalytic domains
•
•
Phosphofructokinase-2 (PFK2) domain catalyzes:
fructose-6-phosphate + ATP ⇄ fructose-2,6-bisphosphate + ADP.
Fructose-Biosphosphatase-2 (FBPase2) domain catalyzes:
fructose-2,6-bisphosphate + H2O ⇄ fructose-6-phosphate + Pi.
Phosphorylation activates FBPase2 and inhibits PFK2
BifunctionalEnzyme
Activates PFK1
Inhibits F-1,6-bisphosphatase
Inhibits PFK1
Activates F-1,6-bisphosphatase
Reciprocal Regulation of PFK-1 and FBPase-1
Medical aside – nonlethal!
People with Type II diabetes have very high (~3x
normal) rates of gluconeogenesis
Initial treatment is usually with metformin.
Metformin shuts down production of PEPCK and
Glucose-6-phosphatase, inhibiting gluconeogenesis.
Futile Cycles
• Occur when loss of reciprocal regulation fails twixt
glycolysis and gluconeogenesis
• Anesthestics like halothane occasionally lead to
runaway cycle between PFK and fructose-1,6-BPase
• Malignant Hyperthermia
The Cori Cycle
Low NADH/NAD+
High NADH/NAD+