Gluconeogenesis (formation of new sugar) 1. Why gluconeogenesis? 2. How different precursors enter gluconeogenesis. 3. The synthesis of glycogen, starch and sucrose; also lactose. 4. The role of sugar-nucleotide.

Gluconeogenesis Gluconeogenesis happens in all animals, plants, and fungi. All the reactions are the same except the regulation. In higher animals, gluconeogenesis happens in liver and renal cortex. Gluconeogenesis can also happen in brain, skeletal and heart muscle. However, liver and kidney remain the main site for this pathway. In liver the major function of gluconeogenesis is to maintain blood glucose. The gluconeogenesis described here is the mammalian pathway.

Why gluconeogenesis? Brain, nervous system, erythrocytes, testes, renal medulla, and embryonic tissues can only utilize glucose from blood as their major or only energy source. Between meals and during longer fasts, or after vigorous exercise, glycogen is depleted. In order to keep the above systems functional, organisms need a method for synthesizing glucose from noncarbohydrate precursors.

Noncarbohydrate precursors for gluconeogenesis Animals Plants Lactate Glycerol Pyruvate Glucogenic amino acids Stored fats Stored proteins Microorganisms Acetate, lactate, propionate

Three reactions are irreversible in glycolysis and must be bypassed during gluconeogenesis Glucose  glucose 6-phosphate (hexokinase) Glucose 6-phosphate  fructose 6-phosphate (phosphohexose isomerase) Fructose 6-phosphate  fructose 1,6-bisphosphate (PFK-1) Fructose 1,6-bisphosphate  dihydroxyacetone phosphate + glyceraldehyde 3-phosphate (aldolase) Dihydroxyacetone phosphate  glyceraldehyde 3-phosphate (triose phosphate isomerase) Glyceraldehyde 3-phosphate  1,3-bisphosphoglycerate (glyceraldehyde 3-phosphate dehydrogenase) 1,3-bisphosphoglycerate  3-phosphoglycerate (phosphoglycerate kinase) 3-phosphoglycerate  2-phosphoglycerate (phosphoglycerate mutase) 2-phosphoglycerate  phosphoenolpyruvate (enolase) Phosphoenolpyruvate  pyruvate (pyruvate kinase) These irreversible reactions will be bypassed in gluconeogenesis.

Reactions in gluconeogenesis Pyruvate  phosphoenolpyruvate Phosphoenolpyruvate  2-phosphoglycerate (enolase) 2-phosphoglycerate  3-phosphoglycerate (phosphoglycerate mutase) 3-phosphoglycerate  1,3-bisphosphoglycerate (phosphoglycerate kinase) 1,3-bisphosphoglycerate  glyceraldehyde 3-phosphate (glyceraldehyde 3-phosphate dehydrogenase) Glyceraldehyde 3-phosphate  dihydroxyacetone phosphate (triose phosphate isomerase) Dihydroxyacetone phosphate + glyceraldehyde 3-phosphate  fructose 1,6-bisphosphate (aldolase) Fructose 1,6-bisphosphate  fructose 6-phosphate (fructose 1,6-bisphosphatase) Fructose 6-phosphate  glucose 6-phosphate (phosphohexose isomerase) Glucose 6-phosphate  glucose (glucose 6-phosphatase)

Three bypasses in gluconeogenesis Because both glycolysis and gluconeogenesis happen in cytosol, reciprocal and coordinated regulation is necessary.

First bypass: from pyruvate to phosphoenolpyruvate (PEP) There are two pathways from pyruvate to PEP. The major pathway uses pyruvate/alanine as glucogenic precursor; however the second pathway will dominate when lactate is the glucogenic precursor. This step involved both cytosolic and mitochondiral enzymes.

Main pathway of the first bypass (1) (mito) The carboxylation of pyruvate by pyruvate carboxylase activates it, initiating the process of gluconeogenesis. Pyruvate carboxylase uses biotin as a carrier of activated HCO3-.

Reaction mechanism of pyruvate carboxylase

Structure of pyruvate carboxylase

Reaction mechanism of pyruvate carboxylase Reaction of pyruvate carboxylase happens in two phases, which occur at two different sites in the enzyme. Biotin is covalently linked to the e-amino group of a Lys residue and acts as a flexible arm between two active sites.

Main pathway of the first bypass (2) Oxaloacetate (OAA) produced by pyruvate carboxylase is then transported out of mitochondria in the form of malate (mitochondrial membrane has no OAA transporter). OAA+NADH+H+  L-malate + NAD+ (mito) (mito) malate dehydrogenase

Main pathway of the first bypass (3) After OAA left mitochondria in the form of malate, it will be converted back to OAA by the cytosolic malate dehydrogenase (p.546, eq. 14-6). This reaction also brings NADH from mitochondria to cytosol, which will help gluconeogenesis to proceed in the latter stage.

Main pathway of the first bypass (4) OAA is then converted to PEP by PEP carboxykinase with GTP as the phosphoryl group donor. The same CO2 that activates pyruvate at the first step is lost during this reaction.

Alternative pathway of first bypass: when lactate is precursor Lactate produced from erythrocytes or anaerobic muscle will be converted to pyruvate first by lactate dehydrogenase (LDH) in hepatocytes (the reverse of lactate fermentation).

Alternative pathway of first bypass (2) Pyruvate is then transported into mitochondria, where it is converted to OAA by pyruvate carboxylase.

Alternative pathway of first bypass (3) OAA is then converted to PEP by mitochondrial PEP carboxykinase (encoded by separate nuclear gene).

Second bypass: conversion of fructose 1,6-bisphosphate to fructose 6-phosphate Because the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate is highly exergonic, the reverse reaction in gluconeogenesis is catalyzed by a different enzyme, FBPase-1.

Third bypass: conversion of glucose 6-phosphate to glucose Similar condition also happened in third bypass. Dephosphorylation of glucose 6-phosphate yielding glucose is catalyzed by glucose 6-phosphatase. However, this reaction does not happen in every tissue.

Third bypass Glucose 6-phosphatase is found on the lumenal side of the ER of hepatocytes and renal cells. It is activated by Mg2+. Muscle and brain tissue do not contain this enzyme and so cannot carry out gluconeogenesis.

Gluconeogenesis is energetically expensive, but essential For glycolysis, every glucose generate 2ATP and 2NADH (p.548). However, 6ATP (4ATP+2GTP) and 2NADH were spent to generate 1 glucose from 2 pyruvate (p.548, eq. 14-9). The extra energy spent is to ensure the irreversibility of gluconeogenesis.

Many amino acids are glucogenic

Pyruvate Alanine Cysteine Glycine Serine Tryptophan Asparagine aspartate Phenylalanine tyrosine Glutamine Arginine Glutamate Histidine proline Isoleucine Methionine Threonine valine

Glycolysis and Gluconeogenesis must be reciprocally regulated ATP + Fructose 6-phosphate  ADP + Fructose 1,6-bisphosphate Fructose 1,6-bisphosphate + H2O  fructose 6-phosphate + Pi ATP + H2O  ADP + Pi + Heat