Glycogen Metabolism andGluconeogenesisCH 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 sugarsGlycogen
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 BreakdownGlycogen synthesis and breakdown are both controlled by hormonesGlucagon, Epinephrine turn on glycogen breakdownTurn off glycogen synthesisHormones act through receptors on cell surface and G-proteins
Glucagon 29 amino acid polypeptide produced in pancreas in response to low blood sugarEpinephrine aka adrenaline produced by adrenal medulla in response to stress
Activation of Glycogen Phosphorylase3-5 cyclic AMPG-ProteinsSecond messengersKinase Cascade
G-ProteinsG proteins are heterotrimers, containing Ga, Gb and Gg subunits.
SubunitSize Ga45 47 kDGb35 kDGg7-9 kD
G-ProteinsThe 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 BindingGTP terminal PO4 constrains the bg-binding loop (red) GDP missing terminal PO4 allows the bg-binding loop (red) to assime a looser conformation
Cycling of G protein between active and inactive states
G-Protein KillersCholera 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 subunit of Gs.Ga is irreversibly modified by addition of ADP-ribosyl group;Modified G 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 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 G 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 PhosphorylaseExists in 2 formsPhosphorylase 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 Phosphorylase3-5 cyclic AMPcAMP dependentProtein Kinase
Activation of Glycogen PhosphorylasecAMP dependentProtein KinasePLP: Pyridoxal Phosphate cofactor
Debranching EnzymeThe 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 DiseaseCori 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 SynthesisGlycogen Synthase adds glucose residues to glycogenSynthase cannot start from scratch needs a primerGlycogenin 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-GlucoseUDP-Glucose Pyrophosphorylase links UDP to glucose
Glycogen Synthesis (cont.)Synthase then adds the activated glucose to the growing chainRelease and subsequent hydrolysis of pyrophosphate drives the reaction to the right
Glycogen Synthesis (cont.)Glycogen branching enzyme then introduces branch points
Mature GlycogenBuilt around glycogenin coreMultiple non-reducing ends accessible to glycogen phosphorylase
Reverse Regulation of Phosphorylase and SynthaseThe same kinase phosphorylates both glycogen phosphorylase and synthaseSynthase I (dephos.) is always activeSynthase D (phos.) is dependent on [G-6-P]The same event that turns one on turns the other one off.
GluconeogenesisAverage adult human uses 120 g/day of glucose, mostly in the brain (75%)About 20g glucose in body fluidsAbout 190 g stored as glycogenLess than 2 days worthIn addition to eating glucose, we also make itMainly occurs in liver (90%) and kidneys (10%)Not the reverse of glycolysisDiffers at the irreversible steps in glycolysis
GluconeogenesisDiffers HereAnd HereAnd Here
First DifferenceGlycolysis: make a nucleotide triphosphateGluconeogenesis: burn two nucleotide triphosphates
Malate ShuttlePyruvate Carboxylase is mitochondrialOAA reduced to malate in matrixCarrier transports malate to cytoplasmCytoplasmic malate dehydrogenase reoxidizes to OAAMammals have a mitochondrial PEPCK
Second and Third differences
EnergeticsGluconeogenesisPyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H2O glucose + 4 ADP + 2 GDP + 2 NAD+G = -37 kJ/molGlycolysis (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 DGs
Local RegulationPhosphofructokinase-1(Glycolysis) is inhibited by ATP and Citrate and stimulated by AMP. Fructose-1,6-bisphosphatase (Gluconeogenesis) is inhibited by AMP.
Global ControlEnzymes 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-BisphosphateFructose-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
BifunctionalEnzymeActivates PFK1Inhibits F-1,6-bisphosphataseInhibits PFK1Activates 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.