Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework** Consortium leader PETER PAZMANY UNIVERSITY Consortium members SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund *** 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 1 **Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben ***A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg. PETER PAZMANY UNIVERSITY SEMMELWEIS UNIVERSITY sote_logo.jpg dk_fejlec.gif INFOBLOKK 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 2 Semmelweis University BIOCHEMISTRY ANABOLISM OF CARBOHYDRATES www.sote.hu BIOKÉMIA A SZÉNHIDRÁTOK SZINTÉZISE TRETTER LÁSZLÓ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATES Table of contents: Gluconeogenesis and its regulation Glycogen synthesis and its regulation Lactose synthesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 3 http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATES Gluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 4 Learning objectives At the end of the presentation students will be able: To demonstrate the difference between the steps of gluconeogenesis and glycolysis. To demonstrate the reactions, specific for gluconeogenesis. To understand the concept of gluconeogenesis i.e. that the higher multicellular organisms use many noncarbohydrate precursors for the biosynthesis of glucose, because the maintenance of blood sugar level is highly important. To understand the principles of metabolic regulation by the comparison the regulation of gluconeogenesis and glycolysis. http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis GluconeogenesisDEF: biosynthesis of glucose from simpler, non-carbohydrate precursors The most important precursors of gluconeogenesis: lactate, pyruvate glycerol (glucogenic) amino acids fatty acids with odd number of carbon atoms Glucogenic amino acidsDEF: amino acids with carbon skeleton that can be used in glucose synthesis during gluconeogenesis (e.g. alanine, aspartate) Antonym: Ketogenic amino acidsDEF: amino acids with carbon skeleton that can be used in ketone body synthesis during starvation (e.g. leucine, lysine) 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 5 http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 6 The principles of gluconeogenesis I. Gluconeogenesis requires reactions from glycolysis, citric acid cycle and a few special reactions The nonequilibrium reactions during glucose catabolism obstruct the simple reversal of glycolysis The nonequilibrium reactions: 1. phosphoenol pyruvate›pyruvate 2. fructose 6-P›fructose 1,6-P2 3. glucose›glucose 6-P http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 7 The principles of gluconeogenesis II. The circumvention of glycolytic reactions 1. Glycolysis: phosphoenol pyruvate›pyruvate Gluconeogenesis:pyruvate›oxaloacetate›phosphoenol pyruvate Pyruvate kinase ADP ATP Pyruvate carboxylase Phosphoenol yruvate carboxykinase CO2 ATP ADP+Pi CO2 GDP GTP http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 8 The principles of gluconeogenesis III. The circumvention of glycolytic reactions However: mitochondria are impermeablefor oxaloacetate but oxaloacetate should be present in the cytosol in order to become a substrate for phosphoenol pyruvate carboxykinase (PEPCK) Problem solved by -reversible oxidoreduction and transamination -reversible transports OA MAL NADH NAD+ Malate dehydrogenase (MDH) MAL OA NAD+NADH OA ASP OA ASP .KG Glut Glut .KG transaminase transaminase Malate dehydrogenase (MDH) transaminasesDEF: enzymes catalyzing the reversible transfer of amino group from an amino acid to an oxo acid resulting a new amino acid and a new oxo acid http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 9 The principles of gluconeogenesis IV. The circumvention of glycolytic reactions 2. Glycolysis: fructose 6-P›fructose 1,6-P2 Gluconeogenesis: Fructose 1,6-P2› Fructose 6-P Phosphofructokinase I ATP ADP H2O Fructose 1,6-P2ase http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 10 The principles of gluconeogenesis V. The circumvention of glycolytic reactions 3. Glycolysis: glucose›glucose 6-P Gluconeogenesis: glucose 6-P›glucose ATP ADP Hexokinase Glucokinase G6T Gl 6-P H2O Pi Gl Gl Pi Gl 6-P ER Glucose 6-P is transported by glucose 6P transporter (G6T) then hydrolized in the ER by glucose 6Pase Gl 6-Pase http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 11 TCA CYCLE OA MALATE Ac-CoA MAL OA OA ASP ASP PEP PYR PYR LACTATE MITO CELL MEMBRANE LACTATE Fr 1,6-P Fr 6-P Gl 6-P Pi ER CELL MEMBRANE Gl Gl TRIOSE PHOSPHATES PYR Fr 1,6P2ase PEPCK PC Gl6Pase Gluconeogenesis in the liver from lactate and pyruvate http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 12 Entry of glycerol into the gluconeogenesis (Liver) Dihydroxyacetone-P CELL MEMBRANE Glycerol Fr 1,6-P Fr 6-P Gl 6-P Pi ER CELL MEMBRANE Gl Gl BLOOD Fr 1,6P2ase Gl6Pase Glycerol Glycerol 3-P Glyceraldehyde 3-P Aldolase B Aldolase A Glycerol kinase ATP ADP Glycerol 3-P dehydrogenase NADH NAD ADIPOSE TISSUE http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 13 Dihydroxyacetone-P CELL MEMBRANE Fructose Fr 1,6-P Fr 6-P Gl 6-P Pi ER CELL MEMBRANE Gl Gl Diet Fr 1,6P2ase Gl6Pase Entry of fructose into the gluconeogenesis (Liver) Fructose Fr 1-P Glyceraldehyde Glyceraldehyde 3-P ATP Triokinase Aldolase B Aldolase B Aldolase A Fructokinase http://semmelweis-egyetem.hu/ CARBOHYDRATES 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 14 www.sote.hu TCA CYCLE OA MALATE Ac-CoA MAL OA OA ASP ASP PEP PYR PYR LACTATE MITO CELL MEMBRANE Fr 1,6-P Fr 6-P Gl 6-P Pi ER CELL MEMBRANE Gl Gl TRIOSE PHOSPHATES PYR Fr 1,6P2ase PEPCK PC Gl6Pase Entry of amino acids into the gluconeogenic pathway ALA AMINO ACIDS .-KG Succ-CoA Fumarate AMINO ACIDS Serine MDH GOT MDH http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATES 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 15 www.sote.hu Dihydroxyacetone-P CELL MEMBRANE Glycerol Fr 1,6-P Fr 6-P Gl 6-P Pi ER CELL MEMBRANE Gl Gl BLOOD Fr 1,6P2ase Gl6Pase Entry of glycerol into the gluconeogenesis (Liver) Glycerol Glycerol 3-P Glyceraldehyde 3-P Aldolase B Aldolase A Glycerol kinase ATP ADP Glycerol 3-P dehydrogenase NADH NAD ADIPOSE TISSUE http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 16 Entry of propionyl-CoAinto the gluconeogenesis (Liver) Fatty acids with odd number of carbon atoms Valine Isoleucine Methionine Cholesterol side chain Propionyl-CoA Succinyl-CoA Succinate Fumarate Malate OA PEPCK PEP Glucose http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 17 Regulation of gluconeogenesis Gene expression Covalent modification Allosteric Enzyme Inducer repressor phosphorylation dephosphorylation activator inhibitor Pyruvate carboxylase Glucocort. Glucagon epinephrine (cAMP) insulin - - - - Phosphoenol pyruvate carboxykinase (PEPCK) Glucocort. Glucagon epinephrine (cAMP) insulin - - - - Fr 1,6-P2ase Glucocort. Glucagon epinephrine (cAMP) insulin - - - Fructose 2,6-P2 AMP Pyruvate kinase Glucocort. Glucagon epinephrine (cAMP) insulin - - Fructose 1,6-P2 ATP, alanine http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 18 Regulation at the PEPCK level The promoterregion of PEPCK gene PEPCK (phosphoenol pyruvate carboxykinase) is the rate limiting stepof gluconeogenezis All of the important hormones in the metabolism can regulate the PEPCK expression http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 19 Regulation at the fr 2,6-P2ase level (liver) GLYCOLYSIS GLUCONEOGENESIS Cell membrane Receptor Glucagon [cAMP]^ PKA^ PFK2 P ˇKinase ˇ activity PFK2 ^Phosphatase^ activity + ˇ[fr2,6-P2] ˇ The elevation of [cAMP] increases the rate of gluconeogensis and decreases the rate of glycolysis in the liver + Fr 1,6-P2 Fr 6-P Fr 1,6-P2 Fr 6-P - X X Glucose http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 20 Regulation of gluconeogenesis I Regulated steps: pyruvate carboxylase PEPCK fr 2,6-P2ase Gl 6-Pase Pyruvate carboxylase is regulated step, it catalyses an irreversible reaction, it is at the beginning of the pathway, but it is not entirely comitted to the gluconeogenesis. Pyruvate carboxylase is also an anaplerotic enzyme of the citric acid cycle. Anaplerotic reactions can not be entirely shut off. AnapleroticDEF: a reaction which can replenish the supply of intermediates in a metabolic pathway, e.g. in the citric acid cycle. PEPCK is a rate limiting, irreversible, heavily regulated step at the initial part of the pathway. It is important to note that neither allosteric nor phosphorylation type of regulation has not been detected on the enzyme. It is regulated exclusively on the gene expression level. Rate limiting stepDEF: The slowest step in a metabolic pathway. Usually it is heavily regulated. E.g. The rate limiting step of glycolysis is the fr 6-P fr1,6-P2transformation. http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 21 Regulation of gluconeogenezis II. Fr 1,6-P2ase is also heavily regulated. This enzyme however, not the main regulatory site of the gluconeogenesis. It is far from the beginning of the pathway. It is particularly interesting that the regulation of PFK1 and the fr 1,6-P2ase is very much coordinated. Those conditions and effectors which stimulate Fr 1,6-P2ase activity inhibit PFK1 activity and vice versa. It is very tempting to say that because PFK1 is the rate-limiting enzyme of the glycolysis, Fr 1,6-P2ase should be the rate-limiting one of the gluconeogenesis, however it is not true. Gl6-Pase is also regulated, but not the rate limiting step either. The reasons for that 1. It is at the very end of the pathway, 2. It is not entirely committed to the gluconeogenesis but playing a role in the glycogenolysis as well. Glycogenolysis is activated earlier than gluconeogenesis, so the enzyme is not suitable for the fine tuning of the gluconeogenesis. http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGluconeogenesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 22 Summary Because of thermodinamic reasons gluconeogenesis is not a simple reversal of glycolysis The irreversible steps of glycolysis should be bypassed The most important gluconeogenic precursors join to the pathway at different levels The regulation of glucogenesis occurs at the irreversible reactions. The most important regulatory site of gluconeogenesis is the PEPCK enzyme http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATES 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 23 Major anabolic and catabolic pathways in glucose metabolism CATABOLIC ANABOLIC http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGlycogen synthesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 24 Learning objectives At the end of the presentation students will be able: To demonstrate the difference between the steps of glycogenesis and glycogenolysis. To demonstrate the reactions, specific for glycogenesis. To understand the principles of simultaneous and opposite regulation of glycogenolysis and glycogenesis. http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGlycogen synthesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 25 Overview of glycogen synthesis Glucose Gl 6-P Gl 1-P UDP-glucose UDP phosphoglucomutase Gl 1-P uridyltransferase Glycogen synthase UTP PPi Glucokinase Hexokinase Glucose (n) Glucose (n+1) H2O Pi+Pi ADP ATP Cost of glycogen synthesis: gluco-, hexokinase1 ATP uridyltransferase 1 UTP Total cost2 ATP equivalent http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGlycogen synthesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 26 Initiation of glycogen synthesis, The role of glycogenin Tyr-OH Tyr-O-(glucose)8 Glycogenin Self-glucosylating Primed glycogenin Glycogenin-Glycogen complex Glycogen synthase and Branching enzyme Tyr-O-(glycogen 8 UDP-gluc 8 UDP n UDP-gluc n UDP Glycogen synthase cannot initiate synthesis without having a primer http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGlycogen synthesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 27 The role of branching enzyme in the synthesis 1 4 4 1 1 4 4 4 1 Glycogen synthase Branching enzyme 7UDP-gl 7UDP http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGlycogen synthesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 28 Regulation of glycogen synthesis I Main regulator: reversible phosphorylation/dephosphorylation Protein kinases cAMP Ca2+ Glycogen synthase a active Glycogen synthase b inactive P Phosphoprotein phosphatase + Pi H2O ATP ADP + + Insulin cAMP + - Gl 6-P + http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGlycogen synthesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 29 Cyclic AMP Secondary messengerDEF: An effector molecule synthesized within the cell in response to an external signal (first messenger) such as a hormone Cyclic AMP (cAMP) is formed from ATP by the adenylate cycle enzyme. First messengers, which could elevate cAMP level are glucagon, adrenalin on beta 1,2 receptors, ACTH etc. cAMP can activate protein kinase-A which can initiate a cascade of phosphorylation http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGlycogen synthesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 30 Regulation of glycogen synthesis II Elevation of [cAMP], or [Ca2+] in the liver indicates the presence of glucagon and adrenaline. Meaning of glucagon: hypoglycemia, glycogen should be mobilized Glycogenesis should be inactivated. Meaning of adrenaline “fight or fly” stress, glycogen should be mobilized, glycogenesis should be inactivated cAMP, Ca2+, activate protein kinases which phosphorylate glycogen synthase Phosphorylated glycogen synthase is inactive –synthesis stopped Phoshatases could remove the phosphorylation (inhibition) of the enzyme. cAMP inhibits the phosphatase, maintains phosphorylation of glycogen synthase (GS), maintains inhibition of the synthesis Insulin stimulates the phosphatase, relieves GS from inhibition, stimulates synthesis Gl 6-P precursor of the glycogen synthesis stimulates the inactive GS. http://semmelweis-egyetem.hu/ 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 31 www.sote.hu Glucose stimulates glycogen synthesis in the liver Increased blood glucose stimulates glycogen synthesis in the liver in an insulin independent manner as well. Glucose binds to phosphorylase a, and glucose-bound phosphorylase is a better substrate for phosphoprotein phosphatase. Phosphorylase acts as a glucose receptor. Glucose promotes inactivation of phosphorylase thus inhibits glycogenolysis. However glycogen synthesis should also be stimulated.Active phosphorylase inhibits dephosphorylation of GS by protein phosphatase. But only phosphorylase a can inhibit this process.Conversion of phosphorylase a to b relieves the inhibition, GS will be dephosphorylated, active, glycogen synthesis could start. http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGlycogen synthesis BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGlycogen synthesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 32 Glucose stimulates glycogen synthesis in the liver PP=phosphoprotein phosphatase http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATESGlycogen synthesis 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 33 Summary As gluconeogenesis is not the reversal of glycolysis, glycogen synthesis is also not the reversal of glycogenolysis. Glycogen synthesis needs energy, 2 high energy phosphate required/glucose incorporated into glycogen. Glycogen synthase is unable to prime “de novo” glycogen synthesis, In order to create a new glycogen molecule, a primer is needed, This primer is the self-catalytic glycogenin protein. The glycogen synthesis is regulated both by reversible phosphorylation/dephosphorylation, both by allosteric effectors. Among the allosteric effectors in the liver, the glucose is the most important. Glucose is able to stimulate glycogen synthesis in the liver in the absence of insulin as well. http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATES 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 34 Lactose synthesis LactoseDEF:The disaccharide of the milk, containing glucose and galactose Lactose synthesis: in lactating mammary gland The enzyme:galactosyltransferasecatalyzed reaction UDP-galactose + N-acetyl-glucosamine › Dgalactosyl-N-acetyl-glucosamine protein attached This enzyme has a role in the glycoprotein synthesis The enzyme does not accept glucose as a galactose acceptor. However, after delivery the specificity of the enzyme changes UDP-galactose + glucose ›lactose + UDP lactose synthase Reason for change in the specificity: lactalbumin: produced by the mammary gland upon hormonal influence. Lactalbumin changes substrate specificity of galactosyltransferase. Lactalbumin-galactosyltransferase complex = lactose synthase GlycoproteinDEF: Proteins containing carbohydrate groups http://semmelweis-egyetem.hu/ Biochemistry: Anabolism of carbohydrates 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 35 Recommended literature Orvosi Biokémia (Ed. Ádám Veronika) Textbook of Biochemistry Ed. Thomas Devlin 5th-7th edition http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATES 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 36 Questions: 1. Which allosteric regulators can regulate both the glycolysis and the gluconeogenesis? 2. How would influence the elevation of cAMP the gluconeogenesis 3. Which reactions require ATP in gluconeogenesis starting from lactate 4. Which enzymes are active in phosphorylated form in the glycogen metabolism. 5. What is the effect of insulin on the gluconeogenesis? http://semmelweis-egyetem.hu/ BIOCHEMISTRY: ANABOLISM OF CARBOHYDRATES 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 37 Questions: Which statements are true for the synthesis of glycogen? 1. Glycogen synthase reaction connects free glucose molecules 2. Glycogen synthase requires ATP for the catalysis 3. During synthesis inorganic phosphate will be incorporated into the glycogen 4. The source of incorporated glucose is UDP-glucose 5. Branches are formed as postsynthetic modifications A:1,2,3,B:2,4C:1,5,D:4,5E:4 Which statements are true for the regulation of glycogen metabolism? 1. Phosphorylation stimulates glycogen phosphorylase activity 2. Phosphorylation decreases the activity of glycogen synthase 3. Phosphorylation increases the activity of glycogen synthase 4. Calcium increases the activity of phosphorylase kinase 5. Glucose decreases the activity of glycogen synthase in liver A:1,2,5,B:1,2,4,C:2,3,5,D:3,4,5 http://semmelweis-egyetem.hu/