Transcript Glycogen Metabolism 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
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
3’-5’ cyclic AMP
G-Proteins
G proteins are heterotrimers,
containing Gα, Gβ and Gγ
subunits.
Subunit
Size
Ga
45 – 47 kD
Gβ
35 kD
Gg
7-9 kD
G-Proteins
• The Gα 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, Gα is bound to the Gβ-Gγ dimer. Gα 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 Gα. The change in Gα
causes it to exchange GDP for GTP. GTP activates Gα, causing it to dissociate from
the Gβ-Gγ dimer. The activated Gα binds and activates an effector molecule.
• Gα also has a slow GTPase activity. Hydrolysis of GTP deactivates Gα, which
reassociates with the Gβ-Gγ 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 allowss the βγbinding loop (red) to
assime a looser
conformation
GTP – terminal PO4
constrains the βγ-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.
Gα 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 (α-1,6 linkages)
Activation of Glycogen Phosphorylase
cAMP – dependent
Protein Kinase
3’-5’ cyclic AMP
Activation of Glycogen Phosphorylase
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
phosphorylase and
synthase
• Synthase A (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
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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
Second and Third differences
Energetics
Gluconeogenesis
• Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H2O ⇌ glucose + 4 ADP + 2 GDP + 2 NAD+
•ΔG = -37 kJ/mol
Glycolysis (reversed)
• Pyruvate + 2 ATP + 2 NADH + 2 H2O ⇌ glucose + 2 ADP + 2 NAD+
•ΔG = +84 kJ/mol
Net difference of 4 nucleotide triphosphate bonds at ~31 kJ
each accounts for difference in ΔGs
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, fructose-2,6bisphosphate.
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 fructose-2,6bisphosphate 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 bi-functional 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
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
High NADH/NAD+
Low NADH/NAD+