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A1.2CM1 Cells, Molecules & Metabolism Lecture 21 (2002) Professor M Schweizer

A1.2CM1 Cells, Molecules & Metabolism Lecture 21 (2002) Professor M Schweizer Refer to chapter 16, Stryer, 5e. Gluconeogenesis. Gluconeogenesis ( reversing glycolysis ... almost! ) occurs mainly in liver . Gluconeogenesis in the kidney is significant under some conditions.

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A1.2CM1 Cells, Molecules & Metabolism Lecture 21 (2002) Professor M Schweizer

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  1. A1.2CM1 Cells, Molecules & Metabolism Lecture 21 (2002) Professor M Schweizer Refer to chapter 16, Stryer, 5e Gluconeogenesis

  2. Gluconeogenesis (reversing glycolysis ... almost!) occurs mainly in liver. Gluconeogenesis in the kidney is significant 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. Hexokinase Phosphofructokinase Pyruvate Kinase. These steps must be bypassed in Gluconeogenesis.

  3. Two Glycolysis reactions are bypassed by simple hydrolysis reactions: Hexokinase (Glycolysis) catalyzes: glucose + ATP  glucose-6-phosphate + ADP Glucose-6-Phosphatase (Gluconeogenesis) catalyzes: glucose-6-phosphate + H2O  glucose + Pi

  4. 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 to the active site (see details:Stryer 5e, p455!)

  5. Phosphofructokinase (Glycolysis) catalyzes: fructose-6-P + ATP  fructose-1,6-bisP + ADP Fructose-1,6-bisphosphatase (Gluconeogenesis) catalyzes: fructose-1,6-bisP + H2O  fructose-6-P + Pi

  6. Bypass of Pyruvate Kinase Pyruvate Kinase (Glycolysis) catalyzes: phosphoenolpyruvate + ADP  pyruvate + ATP For bypass of the Pyruvate Kinase reaction, cleavage of 2 ~P bonds is required. DG for cleavage of one ~P bond of ATP is insufficient to drive synthesis of phosphoenolpyruvate (PEP). PEP has a higher negative DG of phosphate hydrolysis than ATP.

  7. Bypass of Pyruvate Kinase (2 reactions): Pyruvate Carboxylase (Gluconeogenesis) catalyzes: pyruvate+HCO3-+ ATPoxaloacetate +ADP+Pi PEP Carboxykinase (Gluconeogenesis) catalyzes: oxaloacetate + GTP  PEP + GDP + CO2

  8. Contributing to spontaneity of the 2-step process: Free energy of one ~Pbond 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.

  9. Pyruvate Carboxylase uses biotin as prosthetic group. Biotin has a 5-C side chain whose terminal carboxyl is in amide linkage to the e-amino group of an enzyme lysine. The enzyme Biotin Protein Ligase catalyzes attachment of biotin to a particular Lys of Pyruvate Carboxylase, and to specific Lys residues in other enzymes that catalyze carboxylation or decarboxylation reactions.

  10. The biotin & lysine side chains form a long swinging arm that allows the biotin ring to swing back & forth between 2 active sites.

  11. Biotin carboxylation is catalyzed at one active site of Pyruvate Carboxylase. ATP reacts with HCO3- to yield carboxyphosphate. The carboxyl is transferred from this ~P intermediate to N of a ureido group of the biotin ring. Overall: biotin + ATP + HCO3-carboxybiotin + ADP

  12. CO2-biotin-enzyme + H+ CO2 + biotin-E deltaG0’ = -4.7 kcal mol-1

  13. At the other active site of Pyruvate Carboxylase, the activated CO2 is transferred from biotin to pyruvate: carboxybiotin+ pyruvate  biotin + oxaloacetate

  14. Pyruvate carboxylase (the biotin is on a flexible tether allowing to move between the ATP-bicarbonate and pyruvate sites)

  15. PEP Carboxykinase catalyzes GTP-dependent oxaloacetate  PEP. It is thought to proceed in 2 steps: Oxaloacetate is first decarboxylated to yield a pyruvate enolate anion intermediate. This is phosphorylated by phosphate transfer from GTP. A metal ion such as Mn++ is required, in addition to Mg++ associated with the nucleotide substrate.

  16. Pyruvate Carboxylase (pyruvateoxaloactate) is allosterically activated by acetyl CoA. [Oxaloacetate] tends to be limiting for Krebs cycle. When gluconeogenesis is active in liver, oxaloacetate is diverted to form glucose. Oxaloacetate depletion hinders acetyl CoA entry into Krebs Cycle. The increase in [acetyl CoA] activates Pyruvate Carboxylase to make oxaloacetate.

  17. Gluconeogenesis Inputs 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 that are transported to the liver for deamination and conversion to gluconeogenesis inputs. Glycerol, derived from hydrolysis of triacylglycerols in fat cells, is also a significant input to gluconeogenesis.

  18. Summary of Gluconeogenesis Pathway: Gluconeogenesis enzyme names in red. Glycolysis enzyme names in blue.

  19. Glycolysis & Gluconeogenesisareboth spontaneous. If both pathways were simultaneously active in a cell, it would constitute a "futile cycle" that would waste energy. Glycolysis: glucose + 2NAD+ + 2ADP + 2Pi 2pyruvate + 2NADH + 2ATP Gluconeogenesis: 2pyruvate + 2NADH + 4ATP + 2GTP  glucose + 2NAD+ + 4ADP + 2GDP + 6Pi Questions: 1. Glycolysis yields how many ~P? 2. Gluconeogenesis expends how many ~P ? 3. A futile cycle of both pathways would waste how many ~P per cycle ? 2 6 4

  20. To prevent the waste of a futile cycle, Glycolysis & Gluconeogenesis are reciprocally regulated. Local Control includes reciprocal allosteric regulation by adenine nucleotides. This insures that when cellular ATP is high (AMP would then be low), glucose is not degraded to make ATP. It is more useful to the cell under such conditions to store glucose as glycogen. • Phosphofructokinase (Glycolysis) is inhibited by ATP and stimulated by AMP. • Fructose-1,6-bisphosphatase (Gluconeogenesis) is inhibited by AMP.

  21. 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 in liver by cAMP-Dependent Protein Kinase results in inhibition of glycolysis, and stimulation of gluconeogenesis, making glucose available for release to the blood. Enzymes relevant to these pathways phosphorylated by cAMP-Dependent Protein Kinase include: • Pyruvate Kinase, a glycolysis enzyme that is inhibited when phosphorylated. • A bi-functional enzyme that makes and degrades an allosteric regulator, fructose-2,6-bisphosphate.

  22. See Stryer 5e, chapter 16, p457-458

  23. Summary of effects of glucagon-cAMP cascade in liver: • Gluconeogenesis is stimulated. • Glycolysis is inhibited. • Glycogen breakdown is stimulated. • Glycogen synthesis is inhibited. • Free glucose is formed for release to the blood.

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