Lecture 7 - Glycogen Metabolism PDF

Dr. Ardina Grüber Nanyang Technological University School of Biological Sciences Division of Structural Biology and Biochemistry Singapore 637551 email: [email protected]  Synthesis and use of glucose...

Dr. Ardina Grüber Nanyang Technological University School of Biological Sciences Division of Structural Biology and Biochemistry Singapore 637551 email: [email protected]  Synthesis and use of glucose in the human body Brain requires 120 grams/day of glucose out of 160 needed by the entire body ~storage ~ form of glucose Glycogen reserves can provide 190 g and glucose in body fluids is ~20 g; hence, the body contains about one day’s supply When glucose is depleted (fasting or prolonged exercise), glucose must be synthesized from other sources Gluconeogenesis (literally, synthesis of new - glucose from noncarbohydrate when glycoge a precursor) ~ lastate glycerol , a a , depleted / I breakdown of (glycogenolysis) form by muscles when proteins exceeds rate of glycolysis rate of oxidative metabolism © Pearson Education Ltd 2015. Copying permitted for purchasing institution only. Overview of glycogen metabolism Glycogen structure and function Glycogen synthesis – glycogenesis Glycogen degradation – glycogenolysis Regulation of glycogen metabolism by intracellular signaling Human glycogen storage diseases  Glycogen The storage form of glucose in most eukaryotic cells (except plants) is glycogen, a large highly branched polysaccharide consisting of glucose units joined by α-1,4 and α-1,6 glycosidic bonds. striped end: non-reducing end , degradatof glyga branch points anomeric C , branch points , interactso Go from another glucose unit creating, X-1 , 6 glycosidic bond Straight chairs chairs anomeric C interacts w giving traight , , Cp from another glusse unit , creating X-1 , 4 glycosidic bond R.K. Murray, D.A. Botham, Harper’s illustrated biochemistry B glycosidic on as lesdable than & glycosidic bondis  Glycogen Glycogen is principally stored in the cytosol granules of liver and muscle. In mammals, depending on the nutritional state, glycogen account for up to 10 % much greater but bee mass of muscle is of the mass of the liver and 2 % of the mass of muscle. than mass of liver, ~ alyrogen amount is : In liver – The synthesis and breakdown of glycogen is regulated to maintain blood greater in muscle than in glucose levels. liver In muscle - The synthesis and breakdown of glycogen is regulated to meet the energy requirements of the muscle cell. Glycogen granule differences : Liver Cell - size of glysogen granules in liver is 3-4 tires than that in muscles than in muscles -concentrate of glyrogen granules I in liver is higher  Glycogen metabolism The three key enzymes required for reversible degradation and synthesis of glycogen are: units by bral in 1. glycogen phosphorylase ~ degradation,clearesgucose unit to chain only synthesizes 2. glycogen synthase - adds glucose existing , traight chains sond breaks a -1 , 6 glyrsidic 3. branching/debranching enzymes - I X-1 , 6 , Glycogen phosphorylase adds glucose by Glycogen branching and forms branches and glycogen synthase debranching enzymes modify modify glycogen at the glycogen at α-1,6 and α-1,4 nonreducing ends. linkages.  Glycogen metabolism Glycogen degradation and synthesis occurs branchina alyrogen in the cytosol and the substrate for these phosphorylase reaction is the free end of the branching creates branches of non-reduct polymer (nonreducing ends). cleare glucose creates straight long ends I in form of glucose--phosphate chains of glucose units substrate of bonded alyogen synthase by 2-114 ~ bonds glycosidic debranching glycogen enzyme synthesis giving cleare X-1 , 4 in glycodeleath branches & paste it to existing activated chain The large number of branch points in cleave a - 1,6 glycogen results in the generation of multiple nonreducing ends that provide a highly efficient mechanism to quickly 6 release and store glucose. x - 1 , Koolman, Color Atlas of Biochemistry, 2nd edition © 2005 Thieme  Glycogen synthesis - Glycogenesis UDP-glucose pyrophosphorylase I high energy phosphate & activated molecula highly molecule I glucose Glucose 1-phosphate + UTP  UDP-glucose + PPi substrate of glycogen synthase hydrolysis8 PPi released sufficient energy PPi + H2O → 2Pi in this vxth drives I topext to form UDP-glucose Glucose 1-phosphate + UTP + H2O → UDP-glucose + 2Pi Although the reaction is reversible the hydrolysis of the pyrophosphate pushes it to the right. ppi Glycogen synthase, the key regulatory enzyme in glycogen synthesis Glycogen synthase can add chain C from existing glycogen attacks anomeric C , of incoming upp-glucose , creating a-1 , 4 linkage glucosyl residue only if the glucose molecules betw polysaccharide chain already i 2 contains more than four residues. extends - already Glycogen synthesis requires a existing chain glyrogen - primer and this priming 6 to f glasse function is carried out by reside glycogenin.  Nu attack anomeric C Glycogenin starts a new glycogen chain Glycogenin is a homodimer (two identical UDP subunits 37 kDa). N attack displaces Glycogenin catalyzes two distinct reactions: Nu a) Initial attack by the hydroxyl group of Tyr194 on C-1 of the glucosyl moiety of UDP- C4 glucose results in a glucosylated Tyr residue. b) The C-1 of another UDP-glucose anomeric reside molecule is now attacked by the C-4 glycosylated Tyr C hydroxyl group of the terminal glucose. This sequence repeats to form a nascent glycogen molecule of eight glucose residues attached by (14) glycosidic linkages. displaced to f bord => glucose units attached by a-1 , 4 glycosidic  Synthesis of branches in glycogen chains ~ recognise at least 10 to 11 residues that are it long , a clearesX-1 , 4 & paste to glucose unit in at least 4 glucose units from awaycore glycogen remains burried glycogenin ine middle Crystal Structure of Human Glycogen Branching Enzyme (Gbe1) H. M. "Medical gallery of Mikael Häggström 2014”  Glycogen synthase is the key regulatory enzyme in glycogen synthesis Glycogen synthase is found as a homodimer. The activity of glycogen synthase is regulated by covalent modification and allosteric ligand alteration. < phosphorylat bPhosphorylat a Multi-site phosphorylation markedly - changes the net charge of the enzyme at N- and C- terminal ends. The net charge of the enzyme before (green) and after (red) complete phosphorylation. & more - Ve , As P conformati & become inactive  Glycogen synthase is the key regulatory enzyme in glycogen synthesis Enzyme is phosphorylated at multiple sites by glycogen synthase kinase 3 (GSK3), protein kinase A (PKA), casein kinase (CKII) and other kinases. has dual funct stops phosphorylate of GSA - synthase ~ Insulin triggers activation of glycogen b by E ② blocking the activity of GSK3 and activating a phosphoprotein phosphatase (PP1 in muscle, another phosphorylated phosphatase in liver). & dephosphorylate GSB making it active ost as allosteric factor ~ not phosphorylate Glucose 6-phosphate favors dephosphorylation of y glycogen synthase by binding to it and promoting a 1 conformation that is a good substrate for PP1. inhibit PPI , not directly ~ hormone keeping glycogen synthase inactive Glucose also promotes dephosphorylation. & not - liver allosteric factor of glyrogen ( muscles synthase secreted when blood glucose level is too high  Glucose ↑→ Insulin released Glucose ↓→ Glucagon released ↓ ↓ [G] mg/dL Glycogen synthesis Glycogen breakdown Hyperglycemia (eye, nerves, kidney-Diabetes) 126 120 normal range G G 70 40 Hypoglycemia (tired, lethargic, coma, death) EAT time Hormonal regulation of fuel metabolism Maintenance of blood glucose levels is critical to brain function Major hormones: – Insulin – Glucagon -sense glus a – Epinephrine (adrenal gland) & fore muscles instead of glucagon  Hormonal control is hierarchical The pituitary is the first target for most hormones and is under audinag hypothalamic control Pituitary hormones then act on secondary targets Neural stimulation of the adrenal medulla releases epinephrine 7 also known as adrenaline (fight or flight © 2016 Pearson Education, Ltd.  Regulation of glucose metabolism in the liver Gα- G protein cAMP-cyclic AMP PKA-protein kinase A PP-protein phosphatase PDE- phosphodiesterase GSK-3- glycogen synthase ↓ ↑ activate ODE which catalyses kinase 3 converse of CAMP to AMP PI3K – Phosphatidylinositol -3-kinase dual action i also indirectly inhibits PKA inactivatesis - Koolman, Color Atlas of Biochemistry, 2nd edition 2005  How does glucose activate glycogen synthase? allosteric regulation conf - dephosphorylat phorylase A to B active becomes - not active here dephosphorylate (active) Glucose also promotes dephosphorylation; the binding of glucose to glycogen phosphorylase a active site forces a conformational change that favors dephosphorylation to glycogen phosphorylase b, thus allowing the action of PP1.  Glycogen degradation 2 1 - Glycogenolysis Glycogen degradation steps: 3 1. the release of glucose 1- phosphate from glycogen, 2. rearranging the remaining glycogen to permit continued breakdown, and 3. the conversion of glucose 1- phosphate into glucose 6-phosphate for further metabolism.  Glycogen phosphorylase catalyzes starting from non-reducta the breakdown of glycogen Glycogen + Pi  Glucose 1- phosphate + glycogen (n residues) (n-1 residues) - phosphorolytic cleavage is energetically advantageous because released sugar is phosphorylated - equilibrium shifted towards products because of high Pi to Glc-1-P ratio.: favours GP formati  Two additional enzymes are required Step 1: Transferase activity- transfer of 3 Glc residues transferase cleare a- 1 , 4 glycosidic from one outer branch to the y other - cannot act on X-1 , 6 glycosidic bond Step 2: α-1,6-gluosidase activity- & trsf them & paste bond via on hydrolytic release of α(1,6)- glycosidia linked glucose X-1 , 4 alr existf chain cells needs hydrolysis it S a molecule of ATP to cleare a- 1 6 , convert it into Glp straight chain remains , substrate for glyrogen phosphorylate  α-1,6-Glucosidase, debranching enzyme In eukaryotes, the transferase and the α-1,6-glucosidase activities are present in a single 160-kd polypeptide chain, providing yet another example of a bifunctional enzyme. & w2 domains i 2 func?  Phosphoglucomutase converts glucose 1-phosphate into glucose 6-phosphate ~ Ogup interact e & catalyse = treferred to serive cregenerated) phosphorylated transferreda T phosphoglucomutase - positi C, phosphate gup intermediate A phosphoryl group is transferred from the enzyme to the substrate, and a different phosphoryl group is transferred back to restore the enzyme to its initial state. Glucose 6-phosphatase, a hydrolytic enzyme absent from muscle A major function of the liver is to maintain a near constant level of glucose in the blood. The liver contains a hydrolytic enzyme, glucose 6-phosphatase, which cleaves the phosphoryl group to form free glucose and orthophosphate. Glucose 6-phosphatase is absent from most other tissues. Glucose 6 - phosphate + H2O  Glucose + Pi Glucose-6-phosphate is dephosphorylated in the liver for transport out of the liver disrupt active site does not face e cytosol as it will glycolysis GGP be degraded by osphosphatase ~ as will ~ generated into sol transmemb. P active Site facing ER lumen  ~ allosteric regulated Catalytic mechanism of glycogen phosphorylase The special challenge faced by the glycogen phosphorylase is to cleave glycogen phosphorolitically rather than hydrolytically to save the ATP required to phosphorylate free glucose. far but have a crevice big enough to away units, this allows accommodate 4-6 glucose fire catalytic steps do a same E to many wo associate & deassociate from its substrate which is reg for allosteric E like glyrogen phosphorylase slightly biddeaeminal Glycogen phosphorylase (dimer of two identical subunits)  Glycogen phosphorylase requires pyridoxal phosphate (PLP) as a cofactor from glycogen phosphorylase The aldehyde group of this coenzyme forms a Schiff base with a specific lysine side chain of the enzyme. => GP is active The 5’-phosphate group of PLP acts in tandem with ortophosphate by serving as a proton donor and then as a proton acceptor.  Phosphorylase mechanism - estable & attacks phosphate gup non-reducing end crea) & attaches it & C , GIP ordered phosphate donates proton to leaving/remains PLP accepts : glycogen chain proton back to maintain stability , at donor prp donates proton to phosphate grp of acceptor A bound HPO42- group (red) favors the cleavage of the glycosidic bond by donating a proton to the departing glycogen (black). This reaction result in the formation of carbocation and it is favored by the transfer of a proton from the protonated phosphate group of the bound pyridoxal phosphate PLP group (blue). The combination of the carbocation and the orthophosphate results in the formation of glucose 1-phosphate.  Phosphorylase is an allosteric enzyme alsoregulateeath Muscle causeeonylated Active Less active / phosphorylath dephosphorylat covalent modificate - Phosphorylase is regulated by several allosteric effectors that signal the energy state of the cell as well as by reversible phosphorylation, which is responsive to hormones such as insulin, epinephrine, and glucagon.  Regulation of glycogen breakdown: ~ most e active state phosphorylase a and b X-relices ave pulled out The position of the equilibrium of of active site phosphorylase between the T and the R form is responsive to conditions in the cell. PP1 rest The equilibrium for phosphorylase a favors the R state and for phosphorylase b favors the T state. differ in a corf = A Muscle The transition from T state to R state is associated with structural changes in α helices that move a lop out of the acitve site of each subunit. The regulatory enzyme phosphorylase kinase relices tense PP1 areprojea catalyzes covalent modification active site (phosphorylation). - cond" Ybtoa need 2 ATP normal physiological (resting) low activity 1 , > - E Protein phosphatase 1 = PP1 L dephosphorylth , a to be  Allosteric regulation of glycogen breakdown in muscle by AMP, ATP and G6P Muscle phosphorylase b is active only in the presence of high concentrations of AMP, which binds to a nucleotide- binding site and stabilize the conformation of phosphorylase b in the R state. bird to R it ATP acts as a negative allosteric state & brings to T State effector by competing with AMP and so favors the T state. AMP for e The enzyme is inhibited by G6P ATP & are competing side [AMP] same nucleotide-binding , high (feedback inhibition). App binds to e active site , I on other hand when [ATP) ↑, displaces & , structure A, resulting Corfu in A in phosphorylace B as AMP displaced , ATP have diff structures giving convent to i state i low AMP & , level higher activity , & state which is & activity  Muscle glycogen phosphorylase Glycogen phosphorylase has 2 forms: 1. Phosphorylase a: – active form – 2 subunits, in each Ser residue (14) is phosphorylated (Phosphorylase kinase) 2. Phosphorylase b: – inactive form – in resting muscle, all enzyme is its inactive form – structurally identical except that Ser residues are not phosphorylated. It is active when AMP is high! It is inactive when ATP and Glc 6-P are high! – The rate of glycogen breakdown is due to the a/b which is controlled by hormones especially by epinephrine. Phosphorylase a  phosphorylase b by dephosphorylation catalyzed by protein phosphatase 1. once movement of body ↑ hormonal regulate of enzymes which activates phosphorylate , level of phosphorylate b to which has I highest active converting a , , insensitive to allosevic affectors like AMP ALP GGP phosphorylate a is , ,  Liver glycogen phosphorylase Liver phosphorylase and muscle phosphorylase are 90% identical in amino acid sequence. Liver phosphorylase a, but not b, has the most responsive T – to - R transition. ~ in R State The binding of glucose shifts the , A conf relices allosteric equilibrium of the a form from the R to the T state, deactivating the enzyme. Why would glucose function as a negative regulator of liver phosphorylase a? When there is plenty of glucose, no need to breakdown liver glycogen!  Phosphorylase kinase is activated by phosphorylation and calcium ions The phosphorylase kinase enzyme: Has a fully active form and an inactive form Has a mass of 1200 kd very big ~ Consist of 4 subunits (αβγδ) – The subunit γ is the source of catalytic activity. – The other subunits are regulatory subunits. Is under dual control: 1. Regulated by phosphorylation – The β subunit is phosphorylated by cAMP dependent PKA. 2. Partly activated by calcium levels (1 mM). – The δ subunit is calmodulin, a calcium sensor that stimulates many enzymes. Phosphorylase kinase has the highest activity only after both the phosphorylation of the subunit β and the activation of the subunit δ by Ca binding. Phosphorylase kinase  hormones (muscle) River) Cascade mechanism of epinephrine -GTP binding P and glucagon action activates By binding to specific surface receptors, either epinephrine acting on a myocyte (left) or glucagon acting on a hepatocyte (right) activates a GTP- binding protein Gs. which ~ activates adenylyl cyclase Active Gs triggers a rise in [cAMP], activating PKA. This sets off a cascade of phosphorylations; PKA activates phosphorylase b kinase, which then activates glycogen phosphorylase. The resulting breakdown of glycogen provides glucose, which in the myocyte can supply ATP (via glycolysis) for muscle contraction and in the hepatocyte is released into the blood to counter the low blood glucose. 669  Regulation of glucose metabolism in the liver Gα- G protein cAMP-cyclic AMP PKA-protein kinase A PP-protein phosphatase PDE- phosphodiesterase GSK-3- glycogen synthase kinase 3 PI3K – Phosphatidylinositol -3-kinase / inhibitor P remain in active state & start degradat? ~ whe -this is ive inactive Koolman, Color Atlas of Biochemistry, 2nd edition 2005  Glycogen storage diseases of humans absence caused by complete of specific enzymes or malfunc of these enzymes Thank you