Glycogen Metabolism and Gluconeogenesis CH 339 K Glycolysis

Glycogen Metabolism and Gluconeogenesis CH 339 K

Glycolysis (recap) • We discussed the reactions which convert glucose to pyruvate: C 6 H 12 O 6 +2 NAD+ + 2 ADP 2 CH 3 COCOOH + 2 NADH +2 ATP + 2 H+ • What about the sources of glucose? – Dietary sugars – Glycogen

Before we get to glycogen: Dietary sugars

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 k. D Gb 35 k. D Gg 7 -9 k. D

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 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 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 PO 4 allows the bg-binding loop (red) to assime a looser conformation GTP – terminal PO 4 constrains the bg-binding loop (red)

Cycling of G protein between active and inactive states

G-Protein Killers 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 c. AMP 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 c. AMP-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 c. AMP – dependent Protein Kinase 3’-5’ cyclic AMP

Activation of Glycogen Phosphorylase PLP: Pyridoxal Phosphate cofactor c. AMP – 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 339 K

Gluconeogenesis • Average adult human uses 120 g/day of glucose, mostly in the brain (75%) – About 20 g 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

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 H 2 O ⇌ glucose + 4 ADP + 2 GDP + 2 NAD+ • G = -37 k. J/mol Glycolysis (reversed) • Pyruvate + 2 ATP + 2 NADH + 2 H 2 O ⇌ glucose + 2 ADP + 2 NAD+ • G = +84 k. J/mol Net difference of 4 nucleotide triphosphate bonds at ~31 k. J each accounts for difference in DGs

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 c. AMP-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 -Bisphosphate Fructose-2, 6 -bisphosphate is synthesized and degraded by a bifunctional enzyme that includes two catalytic domains • • Phosphofructokinase-2 (PFK 2) domain catalyzes: fructose-6 -phosphate + ATP ⇄ fructose-2, 6 -bisphosphate + ADP. Fructose-Biosphatase-2 (FBPase 2) domain catalyzes: fructose-2, 6 -bisphosphate + H 2 O ⇄ fructose-6 -phosphate + Pi. Phosphorylation activates FBPase 2 and inhibits PFK 2

Bifunctional. Enzyme Activates PFK 1 Inhibits F-1, 6 -bisphosphatase Inhibits PFK 1 Activates F-1, 6 -bisphosphatase

Reciprocal Regulation of PFK-1 and FBPase-1

Medical aside – nonlethal! People with Type II diabetes have very high (~3 x 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+