Regulation of Carbohydrate Metabolism

ANABOLISM OUTLINE: Overview of Photosynthesis Key experiments: Light causes oxygen, which is from water splitting (Hill) NADPH made (Ochoa) Separate from carbohydrate biosynthesis (Rubin & Kamen) Carbohydrate Biosynthesis in Animals precursors Cori cycle Gluconeogenesis reversible steps irreversible steps – four Light Reactions energy in a photon pigments HOW Light absorbing complexes Reaction center Photosystems (PS) PSI – oxygen from water splitting PSII – NADPH Proton Motive Force – ATP Overview of light reactions Carbon Assimilation – Calvin Cycle Stage One – Rubisco Carboxylase Oxygenase Glycolate cycle Stage Two – making sugar Stage Three - remaking Ru 1,5P2 Overview and regulation Calvin cycle connections to biosyn. C4 versus C3 plants Kornberg cycle - glyoxylate energetics 2-steps to PEP mitochondria Pyr carboxylase-biotin PEPCK FBPase G6Pase Glycogen Synthesis UDP-Glc Glycogen synthase Branching Pentose-Phosphate Pathway Oxidative phase Non-oxidative/recycling phase ROS and NADH/NADPH shuttles Regulation of Carbohydrate Metabolism Acetyl-CoA/Pyruvate Pyruvate/PEP F6P/FBP: Fru 2,6P2 Glc/Glc6P Glycogen Anaplerotic reactions Regulation of Carbohydrate Metabolism Catabolism vs. Anabolism 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 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. 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 PDH Complex by acetyl-CoA 3 Regulation of Carbohydrate Metabolism Glycogenolysis versus Glycogen Synthesis Catabolism Glycogen Glycogen Phosphorylase Glycogen Synthase Anabolism Phosphorylase Glycogen Synthase Hexokinase Glucose 6phosphatase Phosphofructokinase-1 Fructose 1,6bisphosphatase Pyruvate Kinase – PEPCK – Pyruvate carboxylase UDP-Glc UDP-glucose pyrophosphorylase Glc 1-P Glycolysis versus Gluconeogenesis 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 Dehydrogenase Complex Acetyl-CoA 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. – 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 • 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 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. – 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 • 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. Regulation of Carbohydrate Metabolism Fructose 2,6-(bis)phosphate (bD-Fru-2,6P2) •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* *6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 5 Regulation of Carbohydrate Metabolism Regulation of Glycolysis and Gluconeogenesis by Fru-2,6P2 • Without Fru 2,6P2, STOP glycolysis GO gluconeogenesis. • With Fru 2,6P2 (130 nM), Go glycolysis. • With Fru 2,6P2 (1300 nM), Stop gluconeogenesis. What controls PFK-2/FBPase-2? Regulation of Carbohydrate Metabolism Regulation of Fru-2,6-P2 Levels Insulin 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 Drop relieves the activation of PFK-1, effectively inhibiting glycolysis. Drop increases activity of FBPase-1, stim ulating gluconeogenesis 6 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 Drop relieves the activation of PFK-1, effectively inhibiting glycolysis. Drop increases activity of FBPase-1, stim ulating gluconeogenesis Regulation of Carbohydrate Metabolism Glycogenolysis versus Glycogen Synthesis Catabolism Glycogen Glycogen Phosphorylase Glycogen Synthase Anabolism Phosphorylase Glycogen Synthase Hexokinase Glucose 6phosphatase Phosphofructokinase-1 Fructose 1,6bisphosphatase Pyruvate Kinase – PEPCK – Pyruvate carboxylase UDP-Glc UDP-glucose pyrophosphorylase Glc 1-P 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 phosphoglucomutase Pyruvate Dehydrogenase Complex 7 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 –Glc activates release/Fru inhibits ⊕ Fructose 1-phosphate Regulation of Carbohydrate Metabolism Glycogenolysis versus Glycogen Synthesis Catabolism Glycogen Glycogen Phosphorylase Glycogen Synthase Anabolism Phosphorylase Glycogen Synthase Hexokinase Glucose 6phosphatase Phosphofructokinase-1 Fructose 1,6bisphosphatase Pyruvate Kinase – PEPCK – Pyruvate carboxylase UDP-Glc UDP-glucose pyrophosphorylase Glc 1-P 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 phosphoglucomutase Pyruvate Dehydrogenase Complex 8 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 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) 9 Regulation of Carbohydrate Metabolism Regulation of Glycogen Synthase •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) • 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 Regulation of Carbohydrate Metabolism Regulation of Glycogen Synthase •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) • 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 10 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 Glc1PàGlc6PàGlc • 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 Anaplerotic Reactions Catabolism Anabolism • We introduced the citric acid cycle as a key catabolic pathway. • It has an equal, if not more important, role in anabolism. – The biosynthesis of biological precursors has to begin with elementary materials – Many, if not most, of these starting points come from the several intermediates in the Kreb’s cycle. – This was first appreciated by Hans Kornberg: how to organisms grow on carbohydrates only? o The term comes from the Greek, to ”fill up” or replenish that without this replenishment, the TCA cycle would grind to a halt o Anaplerotic reactions are critical o Recall Enzyme Kornberg discovered (PEPCK) 11 Anaplerotic Reactions Kornberg Cycle = Glyoxylate Cycle • Was intrigued by the fact that bacteria could grow very effectively on a little ammonium and phosphate salts with acetate (2C) • From these they can synthesize all the components of the cell; DNA, DNA, proteins, membrane lipids, cytochromes, everything….. • How do you build all this from a 2-carbon compound knowing how the Kreb’s cycle works? Dr. Kornberg: Lecture 03.29.17 (31:3235:09) 3.5 min 2 Acetyl-CoA + NAD+ ⇌ Succinate + 2 CoASH + NADH Anaplerotic Reactions • Purine Nucleotide Cycle – was first thought be be part of nucleotide degradation or synthesis – In muscle, its now realized as an important anaplerotic pathway Myoadenylate deaminase deficiency Loss of AMP deaminase-1, the muscle-specific isozyme Causes exercise-induced muscle pain H2O + Aspartate + GTP ⇌ NH4+ + GDP + Pi + Fumarate 12 Anaplerotic Reactions • Intermediates in the citric acid cycle can be used in biosynthetic pathways. • Must replenish the intermediates in order for the cycle and central metabolic pathway to continue. • In animals, these 4-carbon intermediates are formed by carboxylation of 3-carbon precursors. Aspartate + GTP AMP deaminase, adenylosuccinate Pyruvate carboxylase deficiency – an inherited metabolic disorder where anaplerosis is greatly reduced. – What is the problem? – How to treat this disorder? – Other anaplerotic substrates such as the odd-carbon-containing triglyceride triheptanoin are used NH4+ + GDP + Pi + Fumarate synthetase, adenylosuccinate lyase Enzyme Kornberg discovered Muscle ANABOLISM I: Summary What we learned: • Gluconeogenesis, a process by which cells can use a variety of metabolites for the synthesis of glucose • The differences between glycolysis and gluconeogenesis – how they are both made thermodynamically favorable – how they are differentially regulated to avoid a futile cycle • The pentose phosphate pathway, a process by which cells can generate pentose phosphates and NADPH. The pentose phosphates can be regenerated into glucose 6-phosphate, for which NO ATP is required. • living organisms regulate the flux of metabolites through metabolic pathways by: – increasing or decreasing enzyme concentrations – activating or inactivating key enzymes in the pathway • the activity of key enzymes in glycolysis and gluconeogenesis is tightly and coordinately regulated via various activating and inhibiting metabolites (Fru 2,6P2) • glycogen synthesis and degradation is regulated by hormones insulin, epinephrine, and glucagon that report on the levels of glucose in the body • the citric acid cycle plays important anabolic roles in the cell: Anaplerosis • organisms have multiple ways to replenish intermediates that are used in other pathways: Lipid and Nitrogen biosynthesis…....... 13