Gluconeogensis

• Gluconeogenesis is the pathway that converts pyruvate and related three and four carbon compounds to glucose.
• Gluconeogenesis occurs in all animals, plants, fungi, and microorganisms. In mammals, it mainly occurs in the liver.
• Gluconeogenesis and glycolysis share several steps, but they are not identical pathways running in opposite directions.
• There are three glycolysis reactions that are essentially irreversible in vivo and cannot be used in gluconeogenesis. They must be bypassed by exergonic reactions.
• Glycolysis:

  1. Glucose is converted to glucose 6-phosphate by hexokinase and ATP.
  2. Glucose 6-phosphate is converted to fructose 6-phosphate
  3. Fructose 6-phosphate is converted to fructose 1,6-bisphosphate by phosphofructokinase-1 and ATP.
  4. Fructose 1,6-bisphosphate is converted to dihydroxyacetone phosphate and glyceraldehyde 3-phoshate
  5. Dihydroxyacetone phosphate is converted to glyceraldehyde 3-phosphate
  6. Glyceraldehyde 3-phosphate is converted to 1,3-biphosphoglycerate, NADH, and H+ with Pi
  7. 1,3-bisphosphoglycerate is converted to 3-phosphoglycerate and ATP
• Glycolysis:

  8 .3-phosphoglycerate is converted to 2-phoshoglycerate

  9. 2-phosphoglycerate is converted to phosphoenolpyruvate

  10. Phosphoenolpyruvate is converted to pyruvate and ATP by pyruvate kinase.
• Gluconeogenesis:

  1. Pyruvate is converted to oxaloacetate by pyruvate carboxylase and ATP
  2. Oxaloacetate is converted to phosphoenolpyruvate by PEP carboxykinase and GTP.
  3. Phosphoenolpyruvate is converted to 2-phosphoglycerate
  4. 2-phosphoglycerate is converted to 3-phosphoglycerate
  5. 3-phosphoglycerate is converted to 1,3-bisphosphoglycerate by ATP
  6. 1,3-bisphosphoglycerate is converted to glyceraldehyde 3-phosphate, Pi, and NAD+ by NADH and H+.
  7. Glyceraldehyde 3-phosphate and dihydroxyacetone phosphate is converted to fructose 1,6-bisphosphate
• Gluconeogenesis:

  8.Fructose 1,6-bisphosphate is converted to fructose 6-phosphate and Pi by fructose 1,6-bisphosphatase-1 and H2O

  9. Fructose 6-phosphate is converted to glucose 6-phosphate

  10. Glucose 6-phosphate is converted to glucose and Pi by glucose 6-phosphatase and H2O
• Which enzyme is specific to the gluconeogenic pathway?

  • Glucose 6-phosphatase
  • Pyruvate carboxylase
  • PEP carboxykinase
  • Fructose 1,6-bisphosphatase
  • All of the answers are correct

  All of the answers are correct. Although it uses seven of the ten enzymes that also act in glycolysis, gluconeogenesis must bypass three of the most exergonic steps in glycolysis with energetically favorable reactions unique to gluconeogenesis.
• Glycolytic reaction steps:

  1. Glucose + ATP --> glucose 6-phosphate + ADP (-16.7)
  2. Glucose 6-phosphate --> fructose 6-phosphate (1.7)
  3. Fructose 6-phosphate + ATP --> fructose 1,6-bisphosphate + ADP (-14.2)
  4. Fructose 1,6-bisphosphate --> dihydroxyacetone phosphate + glyceraldehyde 3-phosphate (23.8)
  5. Dihydroxyacetone phosphate --> glyceraldehyde 3-phosphate (7.5)
  6. Glyceraldehyde 3-phosphate + Pi + NAD+ --> 1,3-bisphosphoglycerate + NADH + H+ (6.3)
  7. 1,3-bisphophoglycerate + ADP --> 3-phosphoglycerate + ATP (-18.8)
  8. 3-phosphoglycerate --> 2-phosphoglycerate (4.4)
• Glycolytic reaction steps part 2:

  9. 3-phosphoglycerate --> phosphoenolpyruvate + H2O (7.5)

  10. Phosphoenolpyruvate + ADP --> pyruvate + ATP (-31.4)
• The three irreversible steps of glycolysis (glucose to glucose 6-phosphate, fructose 6-phosphate to fructose 1,6-bisphosphate, and phosphoenolpyruvate to pyruvate) are characterized by a large negative delta G.
• The first bypass of gluconeogenesis is the conversion of pyruvate to phosphoenolpyruvate requires two exergonic reactions.
• Pyruvate is transported from the cytosol into the mitochondria or generated from alanine within mitochondria by transamination.
• Pyruvate carboxylase is a mitochondrial enzyme that converts pyruvate to oxaloacetate.

  • Requires the coenzyme biotin
  • Pyruvate + HCO3 + ATP --> oxaloacetate + ADP + Pi
• The role of biotin in the pyruvate carboxylase reaction

  1. In site 1, bicarbonate is converted to CO2 at the expense of ATP. Biotin picks up CO2
  2. Long biotinyl-Lys tether moves CO2 from site 1 to site 2
  3. Biotin delivers CO2 to pyruvate in site 2, forming oxaloacetate
• Which enzyme of gluconeogenesis requires biotin as a coenzyme?

  • PEP carboxykinase
  • Glucose 6-phosphatase
  • Aldolase
  • Pyruvate carboxylase
  • Pyruvate kinase

  Pyruvate carboxylase. Biotin serves as a carrier of activated bicarbonate in the carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase.
• The mitochondrial membrane does not have an oxaloacetate transporter.
• Malate dehydrogenase is a mitochondrial enzyme that uses NADH to reduce oxaloacetate to malate.

  • This reaction is readily reversible under physiological conditions
  • Oxaloacetate + NADH + H+ --> L-malate + NAD+
• Malate leaves the mitochondria through a malate transporter in the inner mitochondrial membrane.
• Reoxidation of malate in the cytosol to oxaloacetate forms NADH.

  • Malate + NAD+ --> oxaloacetate + NADH + H+
• Phosphoenolpyruvate carboxykinase converts oxaloacetate to PEP

  • Requires Mg2+
  • Reversible under intracellular conditions
  • Oxaloacetate + GTP --> PEP + CO2 = GDP
• The overall equation for the first set of bypass reactions is:

  • Pyruvate + ATP + GTP + HCO3 --> PEP + ADP + GDP + Pi + CO2
  • The actual free energy is strongly negative (-25 kJ/mol) making the reaction effectively irreversible.
• When lactate is the glucogenic precursor, a second bypass predominates. Here, oxaloacetate is directly converted to PEP in the mitochondrion by a mitochondrial isozyme of PEP carboxykinase.
• Pyruvate gluconeogenesis:

  1. Pyruvate is moved from the cytosol to the mitochondria
  2. Pyruvate is converted to oxaloacetate, ADP, and Pi by pyruvate carboxylase, CO2, and ATP
  3. Oxalacetate is converted to malate and NAD+ by mitochondrial malate dehydrogenase, NADH, and H+
  4. Malate is transported out of the mitochondria
  5. Malate is converted to oxaloacetate, NADH, and H+ cytosolic malate dehydrogenase and NAD+
  6. Oxaloacetate is converted into PEP, ADP, and CO2 by cytosolic PEP carboxykinase and ATP
• Lactate gluconeogenesis:

  1. Lactate is converted into pyruvate, NADH, and H+ by lactate dehydrogenase and NAD+
  2. Pyruvate is transported into the mitochondria
  3. Pyruvate is converted into oxaloacetate, ADP, and Pi by pyruvate carboxylase, CO2, and ATP
  4. Oxaloacetate is converted into PEP, GDP, and CO2 by mitochondrial PEP carboxykinase and GTP
  5. PEP is transported out of the mitochondria
• When lactate is the glucogenic precursor, mitochondria does not need to export malate. Why?

  • The hepatocytes that metabolize lactate lack mitochondrial malate dehydrogenase
  • The conversation of lactate to pyruvate in the cytosol of hepatocytes produced NADH
  • The pathway from lactate to PEP skips the production of oxaloacetate
  • The pathway from lactate to PEP only requires the expenditure of one high energy phosphate equivalent.

  The conversion of lactate to pyruvate in the cytosol of hepatocytes produces NADH.
• Fructose 1,6-bisphosphatase (FBPase-1) converts fructose 1,6-bisphosphate to fructose 6-phosphate by hydrolysis of the C-1 phosphate

  • Requires Mg2+
  • Essentially irreversible
  • Fructose 1,6-bisphosphate + H2O --> fructose 6-phosphate + Pi
  • delta G'degree = -16.3 kJ/mol
• Glucose 6-phosphatase catalyzes the simple hydrolysis of glucose 6-phosphate to glucose

  • Requires Mg2+
  • Only found in the lumen of the endoplasmic reticulum of hepatocytes, renal cells, and epithelial cells of the small intestine
  • Glucose 6-phosphate + H2O --> glucose + Pi
  • delta G'degree = -13.8 kJ/mol
• What enzyme is not part of gluconeogenesis?

  • Glucose 6-phosphatase
  • PEP carboxykinase
  • Pyruvate decarboxylase
  • Fructose 1,6-bisphosphate

  Pyruvate decarboxylase. Pyruvate decarboxylase catalyzes the first reaction of ethanol fermentation. Pyruvate carboxylase, which has a similar name, converts pyruvate to oxaloacetate in the first bypass reaction in gluconeogenesis/
• Gluconeogenesis is energetically expensive, but essential
• The sum of the biosynthetic reactions leading from pyruvate to free blood glucose is :

  • 2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 4 H2O --> glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+
• Gluconeogenesis:

  1. Pyruvate + HCO3 + ATP --> oxaloacetate + ADP + Pi
  2. Oxaloacetate + GTP --> phosphoenolpyruvate + CO2 + GDP
  3. Phosphoenolpyruvate + H2O --> 2-phosphoglycerate
  4. 2-phosphoglycerate --> 3-phosphoglycerate
  5. 3-phosphoglycerate + ATP --> 1,3-bisphosphoglycerate + ADP
  6. 1,3-bisphosphoglycerate + NADH + H+ --> glyceraldehyde 3-phosphate + NAD+ + Pi
  7. Glyceraldehyde 3-phosphate --> dihydroxyacetone phosphate
  8. Glyceraldehyde 3-phosphate + dihydroxyacetone --> fructose 1,6-bisphosphate
  9. Fructose 1,6-bisphosphate --> fructose6-phosphate + Pi
  10. Fructose 6-phosphate --> glucose 6-phosphate
• Gluconeogenesis part 2:

  11. Glucose 6-phosphate + H2O --> glucose Pi
• How many enzymatic reactions comprise gluconeogenesis?

  • 12
  • 11
  • 10
  • 9
  • 8

  11. The first bypass reaction has two steps.
• Glucogenic amino acids are able to undergo net conversion to glucose. Intermediates of the citric acid cycle also undergo oxidation to oxaloacetate.
• The amino acids that enter at pyruvate are alanine, cysteine, glycine, serine, theronine, and tryptophan.
• The amino acids that enter at alpha-ketoglutarate are arginine, glutamate, glutamine, histidine, and proline.
• The amino acids that enter at succinyl-CoA are isoleucine, methionine, threonine, and valine.
• The amino acids that enter ate fumarate are phenylalanine and tyrosine.