Glycogen Metabolism andGluconeogenesis

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

Maltose

Maltose Glucose

Sucrose

Lactose

Glucose

Fructose

Glucose

Galactose

Pancreatic Amylase

Maltase

Sucrase

Salivary Amylase

Lactase

Glucose Epimerase

Glucose Isomerase

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

3’-5’ cyclic AMP

• G-Proteins• Second messengers• Kinase Cascade

G-Proteins

G proteins are heterotrimers, containing G, G and G subunits.

Subunit Size

Ga 45 – 47 kD

G 35 kD

Gg 7-9 kD

G-Proteins

• The G 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, G is bound to the G-G dimer. G 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 G. The change in G causes it to exchange GDP for GTP. GTP activates G, causing it to dissociate from the G-G dimer. The activated G binds and activates an effector molecule.

• G also has a slow GTPase activity. Hydrolysis of GTP deactivates G, which reassociates with the G-G 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

GTP – terminal PO4 constrains the -binding loop (red)

GDP – missing terminal PO4 allows the -binding loop (red) to assime a looser conformation

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.G 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 problem-2009 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 (-1,6 linkages)

Activation of Glycogen Phosphorylase

3’-5’ cyclic AMP

cAMP – dependentProtein Kinase

Activation of Glycogen Phosphorylase

cAMP – dependentProtein Kinase

PLP: Pyridoxal Phosphate cofactor

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, UDP-Glucose

• 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 non-reducing 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, fructose-2,6-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 fructose-2,6-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-BisphosphateFructose-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

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

High NADH/NAD+ Low NADH/NAD+