Regulation of Carbohydrate Metabolism Lecture

OUTLINE: Introduction and review Transport Glycogenolysis Glycolysis Other sugars Pasteur: Anaerobic vs Aerobic ANABOLISM I: Carbohydrates Carbon Assimilation – Calvin Cycle Stage One – Rubisco Carboxylase Know mechanism Oxygenase Glycolate cycle Stage Two – making sugar Stage Three - remaking Ru 1,5P2 Fermentations Pyruvate Krebs’ Cycle Oxidative Phosphorylation Electron transport Chemiosmotic theory: Phosphorylation Overview and regulation Calvin cycle connections to biosynthesis C4 versus C3 plants Kornberg cycle - glyoxylate Fat Catabolism Fatty acid Catabolism Mobilization from tissues (mostly adipose) Activation of fatty acids Transport; carnitine Oxidation: b-oxidation, 4 steps: Protein Catabolism Amino-Acid Degradation Dealing with the nitrogen; Urea Cycle Dealing with the carbon; Seven Families Nucleic Acid & Nucleotide Degradation PHOTOSYNTHESIS: Overview of Photosynthesis Key experiments: Light Reactions energy in a photon pigments HOW Light absorbing complexes-”red-drop experiment” Reaction center Photosystems (PS) PSII – oxygen from water splitting PSI – NADPH Proton Motive Force – ATP Overview of light reactions Carbohydrate Biosynthesis in Animals precursors Cori cycle Gluconeogenesis reversible steps irreversible steps – four energetics 2-steps to PEP in mitochondria: Pyr carboxylase-biotin & PEPCK FBPase G6Pase Glycogen Synthesis UDP-Glc Glycogen synthase branching Pentose-Phosphate Pathway oxidative-NADPH non-oxidative-Ribose 5-P Regulation of Carbohydrate Metabolism Anaplerotic reactions Regulation of Carbohydrate Metabolism Regulation of Pyruvate Dehydrogenase Complex • Allosteric regulation by energy charge and substrate/product –ADP & pyruvate activates –ATP/NADH & acetyl-CoA inhibit • Regulated by reversible phosphorylation of E1 –phosphorylation: inactive –dephosphorylation: active • PDH kinase and PDH phosphatase are part of mammalian PDH complex. –Kinase is activated by ATP. • high ATP à phosphorylated PDH à less acetyl-CoA made • low ATP à kinase is less active and phosphoprotein phosphatase removes phosphate from PDH à more acetyl-CoA made 1 Catabolism vs. Anabolism Regulation of Carbohydrate Metabolism Gene controlled Glycogenolysis versus Glycogen Synthesis Catabolism Glycogen Glycogen Phosphorylase Glycogen Synthase Anabolism Phosphorylase Glycogen Synthase Hexokinase Glucose 6phosphatase Phosphofructokinase-1 Fructose 1,6bisphosphatase -1 Pyruvate Kinase – PEPCK – Pyruvate carboxylase UDP-Glc Glycolysis versus Gluconeogenesis UDP-glucose pyrophosphorylase Glc 1-P phosphoglucomutase • Regulated enzymes often correspond to points in the pathways that have the same substrate and product, but a different enzyme. • Also where there are junctions. • Can you name those enzymes? Acetyl-CoA Pyruvate Dehydrogenase Complex ✓ Regulation of Carbohydrate Metabolism Regulation of Pyruvate All tissues Kinase • Allosterically activated by fructose-1,6-bisphosphate – increase flow through glycolysis – Feed-forward activation • Allosterically inhibited by signs of abundant energy supply. –ATP –acetyl-CoA and long-chain fatty acids –alanine (enough amino acids) Liver only (under hormonal control) Inactivated by phosphorylation in response to signs of glucose depletion (low bloodglucoseàglucagon) (liver only) Glucose from liver is exported to the brain and other vital organs. This is not the only time we’ll see hormonal control of these pathways. 2 Catabolism vs. Anabolism Regulation of Carbohydrate Metabolism Gene controlled Glycogenolysis versus Glycogen Synthesis Glycogen Synthesis Catabolism Glycogen Glycogen Phosphorylase Glycogen Synthase Anabolism Phosphorylase Glycogen Synthase Hexokinase Glucose 6phosphatase Phosphofructokinase-1 Fructose 1,6bisphosphatase -1 UDP-Glc UDP-glucose pyrophosphorylase Glycolysis versus Gluconeogenesis Glc 1-P phosphoglucomutase • Regulated enzymes often correspond to points in the pathways that have the same substrate and product, but a different enzyme. • Also where there are junctions. • Can you name those enzymes? Pyruvate Kinase Pyruvate Dehydrogenase Complex Acetyl-CoA ✓ ✓ – PEPCK – Pyruvate carboxylase Regulation of Carbohydrate Metabolism Regulation of Pyruvate Carboxylase • Allosteric activation of pyruvate carboxylase by acetyl-CoA – stimulates glucose synthesis via gluconeogenesis because plenty of acetyl-CoA signals plenty of CAC intermediates • Notice the reciprocal control of PC & PDH Complex by acetyl-CoA 3 Regulation of Carbohydrate Metabolism Gene controlled Glycogenolysis versus Glycogen Synthesis Catabolism Glycogen Glycogen Phosphorylase Glycogen Synthase Anabolism Phosphorylase Glycogen Synthase Hexokinase Glucose 6phosphatase Phosphofructokinase-1 Fructose 1,6bisphosphatase UDP-Glc Glycogen Synthesis UDP-glucose pyrophosphorylase Glycolysis versus Gluconeogenesis Glc 1-P phosphoglucomutase • Regulated enzymes often correspond to points in the pathways that have the same substrate and product, but a different enzyme. • Can you name those enzymes? Pyruvate Kinase Pyruvate Dehydrogenase Complex Acetyl-CoA ✓ ✓ – PEPCK – Pyruvate carboxylase ✓ Regulation of Carbohydrate Metabolism Regulation of Phosphofructokinase-1 versus Fructose 1,6-bisphosphatase-1 High [AMP] Low [AMP] Homeostatic level of Fru 6-P • Fructose-6-phosphate à fructose 1,6-bisphosphate is the commitment step in glycolysis. • While ATP is a substrate, ATP is also a negative effector. Fructose 2,6bisphosphate – Do not spend glucose in glycolysis if there is plenty of ATP. – Same for citrate, if there is plenty of citrate, do not waste glucose Fructose 2,6bisphosphate ⊕ • Low energy charge inhibits biosynthesis of Glc. Is this a typo? Bumble bees are missing an FBPase that responds to AMP • • Go glycolysis if AMP is high and ATP is low. Go gluconeogenesis if AMP is low. 4 Regulation of Carbohydrate Metabolism Fructose 2,6-(bis)phosphate (bD-Fru-2,6P2) 6 2 5 4 3 1 •NOT a glycolytic intermediate, only a regulator •Produced specifically to regulate glycolysis and gluconeogenesis bD-Fru-1,6P2 – activates phosphofructokinase-1(PFK-1) (glycolysis) – inhibits fructose 1,6-bisphosphatase (FBPase-1) (gluconeogenesis) Enzyme for synthesis and degradation of Fru 2,6P2 done with a dual-function enzyme: PFK-2/FBPase-2* Regulation of Carbohydrate Metabolism Regulation of Glycolysis and Gluconeogenesis by Fru-2,6P2 Homeostatic level of Fru 6-P Low Glc High Glc • Without Fru 2,6P2, STOP glycolysis GO gluconeogenesis. This would be the state of low Glc What controls PFK-2/FBPase-2? Homeostatic level of Fru 1,6-P2 Low Glc High Glc • With Fru 2,6P2 (130 nM), Go glycolysis. • With Fru 2,6P2 (1300 nM), Stop gluconeogenesis. This would be the state of high Glc 5 Regulation of Carbohydrate Metabolism Regulation of Fru-2,6-P2 Levels Structurally, this enzyme, with its two activities is different than those in glycolysis and gluconeogenesis (i.e., they are conjoined, rather than independent) and are regulated via phosphorylation. Glucagon Insulin (PPI) Drop relieves the activation of PFK-1, effectively inhibiting glycolysis. Drop increases activity of FBPase-1, stim ulating gluconeogenesis Regulation of Carbohydrate Metabolism Gene controlled Glycogenolysis versus Glycogen Synthesis Catabolism Glycogen Glycogen Phosphorylase Glycogen Synthase Anabolism Phosphorylase Glycogen Synthase Hexokinase Glucose 6phosphatase UDP-Glc Glycolysis versus Gluconeogenesis • Regulated enzymes often correspond to points in the pathways that have the same substrate and product, but a different enzyme. • Can you name those enzymes? Acetyl-CoA UDP-glucose pyrophosphorylase Glc 1-P phosphoglucomutase Phosphofructokinase-1 ✓ Pyruvate Kinase ✓ Pyruvate Dehydrogenase Complex ✓ ✓ Fructose 1,6bisphosphatase – PEPCK – Pyruvate carboxylase ✓ 6 Regulation of Carbohydrate Metabolism There Are Four Isozymes of Hexokinase (I-IV) Regulation of Hexokinase •Isozymes are different enzymes that catalyze the same reaction. –typically share similar sequences –may have different kinetic properties –can be regulated differently HK-I versus HK-IV •HK I is expressed in all tissues, to different levels. •HK IV (glucokinase) is only expressed in the liver and pancreas. Glucokinase Is Regulated by Sequestration –has higher Km, so responsive to higher [glucose] –not inhibited by glucose-6-phosphate, so can function at higher [glucose] –functions to clear blood glucose at higher [glucose] for storage as glycogen also Fru1P –Glc activates release/Fru6P inhibits ⊕ Fructose 1-phosphate Regulation of Carbohydrate Metabolism Gene controlled Glycogen Synthesis Glycogenolysis versus Glycogen Synthesis Catabolism Glycogen Glycogen Phosphorylase Glycogen Synthase Anabolism Phosphorylase Glycogen Synthase UDP-Glc Glycolysis versus Gluconeogenesis • Regulated enzymes often correspond to points in the pathways that have the same substrate and product, but a different enzyme. • Can you name those enzymes? Acetyl-CoA UDP-glucose pyrophosphorylase Glc 1-P phosphoglucomutase Hexokinase ✓ Phosphofructokinase-1 ✓ Pyruvate Kinase ✓ Pyruvate Dehydrogenase Complex ✓ Glucose 6phosphatase ✓ Fructose 1,6bisphosphatase – PEPCK – Pyruvate carboxylase ✓ 7 Regulation of Carbohydrate Metabolism Glc Glc Regulation of Glycogen Phosphorylase Nobel Prize 1972 Earl Sutherland 1915-1974 •Glycogen phosphorylase cleaves glucose residues off glycogen, generating glucose-1phosphate (Glc 1P). • Phosphorylation activates glycogen phosphorylase-b AMP AMP –Phosphorylase-b Kinase –Accentuated by allosteric binding of AMP (muscle only) • Dephosphorylation inhibits glycogen phosphorylase-a –Phosphoprotein phosphatase-1 (PP1) –Accentuated by allosteric binding of Glc (in liver only) Regulation of Carbohydrate Metabolism Regulation of Glycogen Synthase Need energy: stop storing & release •Glycogen synthase adds glucose residues to glycogen using UDP-Glc. • Phosphorylation inhibits glycogen synthase-a –Its complicated, responding to multiple signals –Example: First Casein Kinase-2 (CKII), then Glycogen Synthase Kinase-3 (GSK3) [inactivated by insulin] • Dephosphorylation activates glycogen synthase-b –Phosphoprotein phosphatase-1 (PP1) (in liver it’s a different PP) –PP1 is bound to GS-b • Also, feedforward control by glucose and Glc-6P –Binding causes a conformation favorable for PP-1 binding –Binding does not allow GSK-3 access to phosphorylation sites Regulation of glycogen synthase/glycogen phosphorylase is somewhat similar to regulation of PDH complex 8 Regulation of Carbohydrate Metabolism Glycogen Phosphorylase Cascade Nobel Prize 1992 Edwin Krebs 1918-2009 & Hepatocyte glucagon receptor b-adenoreceptor G-protein activation •Glucagon/epinephrine signaling pathway activated when there is a NEED for energy – – – – – starts phosphorylation cascade via cAMP cAMP activates PKA PKA activates phosphorylase-b kinase this kinase activates glycogen phosphorylase Massive degradation of glycogen • In muscle: Glc1PàGlc6Pàglycolysis • In liver: Glc1PàGlc6PàBlood glucose • There is reciprocal inhibition of GS – PKA phosphorylates GM , which is bound to PP1 on GS, thus dissociating it. – PKA also phosphorylates PP1-inhibitor protein, which binds and inactivates the free PP1, thus leaving GS-℗ and inactive G M is the glycogentargeting protein Regulation of Carbohydrate Metabolism Gene controlled Glycogenolysis versus Glycogen Synthesis Catabolism Glycogen Glycogen Phosphorylase Glycogen Synthase Anabolism Phosphorylase ✓ Hexokinase ✓ Phosphofructokinase-1 ✓ Pyruvate Kinase ✓ Glycogen Synthase ✓ UDP-Glc Glycolysis versus Gluconeogenesis • Regulated enzymes often correspond to points in the pathways that have the same substrate and product, but a different enzyme. • Can you name those enzymes? Acetyl-CoA UDP-glucose pyrophosphorylase Glc 1-P phosphoglucomutase Pyruvate Dehydrogenase Complex ✓ Glucose 6phosphatase ✓ Fructose 1,6bisphosphatase – PEPCK – Pyruvate carboxylase ✓ 9