Glyconeogenesis.pdf

Gluconeogenesis The process of conversion of lactate to glucose is called gluconeogenesis, uses some of the reactions of glycolysis (but in the reverse direction) and some reactions unique to this pathway to re-synthesize glucose. This pathway requires an ene...

Gluconeogenesis The process of conversion of lactate to glucose is called gluconeogenesis, uses some of the reactions of glycolysis (but in the reverse direction) and some reactions unique to this pathway to re-synthesize glucose. This pathway requires an energy input (as ATP) but has the role of maintaining a circulating glucose concentration in the bloodstream (even in the absence of dietary supply) and also maintaining a glucose supply to fast twitch muscle fibres. Gluconeogenesis occurs mainly in liver. Gluconeogenesis occurs to a more limited extent in kidney & small intestine under some conditions. Synthesis of glucose from pyruvate utilizes many of the same enzymes as Glycolysis. Three Glycolysis reactions have such a large negative DG that they are essentially irreversible. w Hexokinase (or Glucokinase) w Phospho-fructokinase w Pyruvate Kinase. These steps must be bypassed in Gluconeogenesis. Two of the bypass reactions involve simple hydrolysis reactions.  Glucose-6-phosphatase 6 CH OPO 2- CH2OH 2 3 5 O O H H H H H H2O H 4 OH H 1 OH H + Pi OH OH OH OH 3 2 H OH H OH glucose-6-phosphate glucose Hexokinase or Glucokinase (Glycolysis) catalyzes: glucose + ATP à glucose-6-phosphate + ADP Glucose-6-Phosphatase (Gluconeogenesis) catalyzes: glucose-6-phosphate + H2O à glucose + Pi  Glucose-6-phosphatase 6 CH OPO 2- CH2OH 2 3 5 O O H H H H H H2O H 4 OH H 1 OH H + Pi OH OH OH OH 3 2 H OH H OH glucose-6-phosphate glucose Glucose-6-phosphatase enzyme is embedded in the endoplasmic reticulum (ER) membrane in liver cells. The catalytic site is found to be exposed to the ER lumen. Another subunit may function as a translocase, providing access of substrate G-6-P to the active site on G-6-Pase.  Phosphofructokinase ® 6 CH OPO 2- 1CH2OH 6 CH OPO 2- 1CH2OPO32- 2 3 2 3 O ATP ADP O 5 H HO 2 5 H HO 2 H 4 3 OH H 4 3 OH Pi H2O OH H OH H fructose-6-phosphate fructose-1,6-bisphosphate ¬ Fructose-1,6-bisphosphatase Phosphofructokinase (Glycolysis) catalyzes: fructose-6-P + ATP à fructose-1,6-bisP + ADP Fructose-1,6-bisphosphatase (Gluconeogenesis) catalyzes: fructose-1,6-bisP + H2O à fructose-6-P + Pi Bypass of Pyruvate Kinase: Pyruvate Kinase (last step of Glycolysis) catalyzes: phospho-enolpyruvate + ADP à pyruvate + ATP For bypass of the Pyruvate Kinase reaction, cleavage of 2 ~P bonds is required. w DG for cleavage of one ~P bond of ATP is insufficient to drive synthesis of phospho-enolpyruvate (PEP). w PEP has a higher negative DG of phosphate hydrolysis than ATP.  O O- Pyruvate Carboxylase C PEP Carboxykinase - O O O O- C ATP ADP + Pi C O GTP GDP C C O CH2 C OPO32- CH3 HCO3- C CO2 CH2 O O- pyruvate oxalo-acetate PEP Bypass of Pyruvate Kinase (2 enzymes): Pyruvate Carboxylase (Gluconeogenesis) catalyzes: pyruvate + HCO3- + ATP à oxalo-acetate + ADP + Pi PEP Carboxykinase (Gluconeogenesis) catalyzes: oxalo-acetate + GTP à PEP + GDP + CO2  O O- Pyruvate Carboxylase C PEP Carboxykinase - O O O O- C ATP ADP + Pi C O GTP GDP C C O CH2 C OPO32- CH3 HCO3- C CO2 CH2 O O- pyruvate oxalo-acetate PEP Contributing to spontaneity of the 2-step process: Free energy of one ~P bond of ATP is conserved in the carboxylation reaction. Spontaneous decarboxylation contributes to spontaneity of the 2nd reaction. Cleavage of a second ~P bond of GTP also contributes to driving synthesis of PEP. Pyruvate Glucose-6-phosphatase glucose-6-P glucose Carboxylase (pyruvate à oxalo- Gluconeogenesis Glycolysis acetate pyruvate intermediate) is fatty acids allosterically acetyl CoA ketone bodies activated by acetyl CoA. Thus, oxaloacetate citrate [Oxaloacetate] tends to be limiting for Krebs Cycle Krebs cycle. When gluco-neogenesis is active in liver, oxalo-acetate is diverted to form glucose. Oxalo-acetate depletion hinders acetyl CoA entry into Krebs Cycle. The increase in [Acetyl CoA] activates Pyruvate Carboxylase to make oxaloacetate.  The source of pyruvate and oxaloacetate for gluconeogenesis during fasting or carbohydrate starvation is mainly amino acid catabolism. Some amino acids are catabolized to pyruvate, oxaloacetate, or precursors of these. Muscle proteins may break down to supply amino acids. These are transported to liver where they are deaminated and converted to gluconeogenesis inputs. Glycerol, derived from hydrolysis of triacylglycerols in fat cells, is also a significant input to gluconeogenesis.  glyceraldehyde-3-phosphate NAD+ + Pi Glyceraldehyde-3-phosphate NADH + H+ Dehydrogenase 1,3-bisphosphoglycerate ADP Summary of Phosphoglycerate Kinase ATP Gluconeogenesis 3-phosphoglycerate Pathway: Phosphoglycerate Mutase 2-phosphoglycerate Gluconeogenesis enzyme names in H2O Enolase red. phosphoenolpyruvate CO2 + GDP Glycolysis enzyme PEP Carboxykinase GTP names in blue. oxaloacetate Pi + ADP Pyruvate Carboxylase HCO3- + ATP pyruvate Gluconeogenesis  glucose Gluconeogenesis Pi Glucose-6-phosphatase H2O glucose-6-phosphate Phosphoglucose Isomerase fructose-6-phosphate Pi Fructose-1,6-bisphosphatase H2O fructose-1,6-bisphosphate Aldolase glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate Triosephosphate Isomerase (continued) Glycolysis & Gluconeogenesis are both spontaneous. If both pathways were simultaneously active in a cell, it would constitute a "futile cycle" that would waste energy. Glycolysis: glucose + 2 NAD+ + 2 ADP + 2 Pi à 2 pyruvate + 2 NADH + 2 ATP Gluconeogenesis: 2 pyruvate + 2 NADH + 4 ATP + 2 GTP à glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi Questions: 1. Glycolysis yields how many ~P ? 2 2. Gluconeogenesis expends how many ~P ? 6 3. A futile cycle of both pathways would waste how many ~P per cycle ? 4  Phosphofructokinase ® 6 CH OPO 2- 1CH2OH 6 CH OPO 2- 1CH2OPO32- 2 3 2 3 O ATP ADP O 5 H HO 2 5 H HO 2 H 4 3 OH H 4 3 OH Pi H2O OH H OH H fructose-6-phosphate fructose-1,6-bisphosphate ¬ Fructose-1,6-biosphosphatase To prevent the waste of a futile cycle, Glycolysis & Gluconeogenesis are reciprocally regulated. Local Control includes reciprocal allosteric regulation by adenine nucleotides. w Phosphofructokinase (Glycolysis) is inhibited by ATP and stimulated by AMP. w Fructose-1,6-bisphosphatase (Gluconeogenesis) is inhibited by AMP. The opposite effects of adenine nucleotides on w Phosphofructokinase (Glycolysis) w Fructose-1,6-bisphosphatase (Gluconeogenesis) insures that when cellular ATP is high (AMP would then be low), glucose is not degraded to make ATP. When ATP is high it is more useful to the cell to store glucose as glycogen. When ATP is low (AMP would then be high), the cell does not expend energy in synthesizing glucose. Global Control in liver cells includes reciprocal effects of a cyclic AMP cascade, triggered by the hormone glucagon when blood glucose is low. Phosphorylation of enzymes & regulatory proteins in liver by Protein Kinase A (cAMP Dependent Protein Kinase) results in w inhibition of glycolysis w stimulation of gluconeogenesis, making glucose available for release to the blood. Enzymes relevant to these pathways that are phosphorylated by Protein Kinase A include: w Pyruvate Kinase, a glycolysis enzyme that is inhibited when phosphorylated. w CREB (cAMP response element binding protein) which activates, through other factors, transcription of the gene for PEP Carboxykinase, leading to increased gluconeogenesis. w A bi-functional enzyme that makes and degrades an allosteric regulator, fructose-2,6-bisphosphate. Reciprocal regulation by fructose-2,6-bisphosphate (F-2,6-P) stimulates Glycolysis. F-2,6-P allosterically activates the Glycolysis enzyme Phosphofructokinase. Activates transcription of the gene for Glucokinase, the liver variant of Hexokinase that phosphorylates glucose to glucose-6-phosphate, the input to Glycolysis. Allosterically inhibits the gluconeogenesis enzyme Fructose-1,6-bisphosphatase.  CORI CYCLE explains how glucose can be consumed by muscles, leaching lactate in the process. The liver then uses this lactate to create glucose. Muscles normally combine glucose with oxygen to generate energy. If oxygen is unavailable, the anaerobic breakdown of glucose/glycolysis/fermentation results in lactate production lactate (soluble milk acid ) excreted back into the bloodstream Gluconeogenesis (liver) maintains the proper blood sugar level through the synthesis of glucose from non-carbohydrate components. Critical to completing this loop is the catalytic co- enzyme adenosine triphosphate (ATP) Under normal oxygen, glycolysis in muscle cells produces two units of ATP and two units of pyruvate The two compounds provide the energy that enables a cell to perpetuate respiration through a series of chemical reactions called the Krebs cycle, also called the citric acid or tricarboxylic acid cycle.