Gluconeogenesis BIOCHEM 4511 Biological role Energetic balance Reaction sequence Regulation Figures Essential Biochemistry 3rd Ed Pratt and Cornely Principles of Biochemistry 5th Ed Moran et al Biochemistry A short course Tymoszko et al Carbohydrate Metabolism Overview Glycogen 5 Pentose Glucose 1 2ADP 2NAD 2ATP 2NADH H Pyruvate 2 Lactate 4 7 Acetyl CoA 9 ATP Other sugars 6 8 10 TCA Cycle 3 EtOH CO2 1 Glycolysis 1 2 Anaerobic glycolysis 1 3 Alcoholic fermentation 1 4 9 10 Aerobic glycolysis 5 Pentosephosphate shunt 6 Glycogenolysis 7 Gluconeogenesis 8 Glycogenesis 9 TCA Cycle 2 10 Electron Transport Oxidative Phosphorylation Stages of Glucose Homeostasis Brain and RBC almost entirely depend on glucose as fuel Brain can also use ketone bodies under fasting Glucose blood is maintained at nearly constant level of 4 5mM Early on fasting glycogen breakdown can provide glucose Later only GLUCONEOGENESIS can provide glucose Gluconeogenesis de novo formation of glucose from noncarbohydrate metabolites to satisfy energetic brain RBC 3 muscle and metabolic needs of the organism Cori Lactic acid Cycle Gluconeogenesis I Fasting II To compensate for glucose breakdown by anaerobic glycolysis to lactate in actively working muscle and RBC Lactate dehydrogenase Cori Cycle 1 couples fast glucose consumption in muscle and RBC and glucose regeneration in liver 2 regenerates NAD for glycolysis 4 Alanine Cycle Alanine Cycle 1 Couples glucose consumption in muscle and glucose regeneration in liver Transamination reaction Alanine aminotransferase 2 Transports NH3 to liver for utilization energy consuming 3 Does not affect NADH level NADH can be used for aerobic generation of ATP 5 Gluconeogenesis I Tissue organ compartmentation Liver 90 Kidney 10 Some in the intestine II Cellular compartmentation First reaction in mitochondrion the last reaction in ER the rest in the cytoplasm Balance 6 Gluconeogenesis energetic balance Balance 7 Conversion of pyruvate to glucose consumes 6 ATP equivalents If run in cycle with glycolysis gluconeogenesis would consume 4 ATP molecules energetically wasteful Therefore both pathways don t happen simultaneously but adjust to metabolic needs of the cell organism Substrates of gluconeogenesis I Pyruvate oxaloacetate II Lactate pyruvate oxaloacetate III Alanine pyruvate oxaloacetate IV All other amino acids but Leucine and Lysine oxaloacetate V Glycerol dihydroxyacetone phosphate DHAP Not substrates Fatty acids Acetyl CoA Leucine Lysine 8 Gluconeogenesis Glycolysis 3 irreversible steps of Glycolysis are replaced with 4 irreversible steps of Gluconeogenesis 3 in bacteria All 7 near equillibrium reversible reactions are the same 9 Gluconeogenesis Glycolysis 10 Pyruvate is Converted to PEP in Two Steps The conversion from pyruvate to PEP is an endergonic reaction and is coupled with the hydrolysis of both ATP and GTP CO2 is added in the first step and released in the second Step 1 Pyruvate carboxylase Carboxylation of pyruvate to oxaloacetate with biotin prosthetic group as a cofactor 12 Biotin carboxyl group transfer Recognize Biotin is synthesized by intestinal bacteria so its deficiency is rare but can occur upon ingestion of raw eggs that contain protein avidin Biotin Avidin is among the strongest non covalent interactions in nature KD 10 15 M Is biotin a co substrate or a prosthetic group 13 Pyruvate carboxylase summary Uses biotin prosthetic group as a cofactor for carboxylation Carboxylation involves bicarbonate HCO3 Enzyme is mitochondrial in eukaryotes Product oxaloacetate intermediate in Krebs cycle Enzyme is allosterically activated by acetyl CoA produced from pyruvate to enter the Krebs cycle for energy generation Activation by the product of opposing pathway 14 2nd Unique reaction Phosphoenolpyruvate carboxykinase PEPCK Bacteria Animals No allosteric regulation Enzyme is regulated at the level of transcription synthesis degradation Glucagon cAMP PKA increase in PEPCK hormonal induction 15 Insulin reverse Phosphoglycerate kinase Phosphoglycerate mutase enolase GLYCOLYSIS GLUCONEOGENESIS Next six reactions are reversed to those in glycolysis 16 Glucose 6P isomerase phosphogluco isomerase or phosphohexose isomerase Aldolase Triose phosphate isomerase GAP dehydrogenase 17 The Final Two Unique Enzymes of Gluconeogenesis are Phosphatases 3rd unique step Fructose 1 6 bisphosphatase Negative allosteric regulation by AMP Fructose 2 6 Bisphosphate Reciprocal to regulation of Phosphofructokinase 1 PFK1 Which allosteric regulators of PFK1 do not affect F1 6 BP 19 Glucose 6 phosphatase 4th irreversible step Restricted to liver kidney small intestine organs of gluconeogenesis Used to release glucose for other tissues Not present in muscle so muscle glycogen is not used by the rest of the body Enzyme is localized at the luminal side if ER endoplasmic reticulum membrane Overview of Gluconeogenesis Gluconeogenesis is the formation of glucose from pyruvate glycolysis or oxaloacetate TCA cycle Glycolysis enzymes which catalyze reversible reactions are used Four new enzymes are required Pyruvate carboxylase Phosphoenolpyruvate carboxykinase Fructose bisphosphatase Glucose 6 phosphatase Reciprocal Regulation of glycolysis and gluconeogenesis 22 Regulation of Metabolic Pathways Product Inhibition The final product in a metabolic pathway commonly inhibits an enzyme allosterically earlier in the pathway to prevent product accumulation Logically if there are already sufficient quantities of the final product of a pathway there is no need to invest energy and cellular resources to generate additional product Regulation of Metabolic Pathways Feed Forward Activation An early product in a metabolic pathway after the committed step commonly activates an enzyme allosterically later in the pathway to increase final product formation Logically if there is a backlog of an intermediate product there is a need to invest energy and cellular resources to increase the rate of the overall pathway Regulation of Glucose synthesis and breakdown Reciprocal regulation Synthesis glucagon Degradation insulin 25 Role of Fructose 2 6 bisphospate in glucose metabolism Bifunctional regulatory enzyme PFK2 FBP2 1 The abundance of Glucose 6P leads to high level of Fructose 2 6 bisphosphate which activates PFK 1 and inhibits FBPase 1 2 Fructose 2 6 bisphosphate is produced from Fructose 6 phosphate by PFK2 3 Fructose 2 6 bisphosphate is broken down to F6Phosphate by FBPase 2 4 In liver PFK 2 is activated under