Download Biosynthesis of glucose – gluconeogenesis

Biosynthesis of
glucose –
gluconeogenesis
Carbohydrates provide
a significant portion of
human caloric intake
All cells are dependent on glucose.
Glucose level in blood plasma must be stable.
Brain is especially sensitive to the decrease of
glucose level (the daily glucose requirement of the
brain in a typical adult human being is about 120 g).
Red blood cells use only glucose as a fuel.
160 g of glucose needed daily by the whole body.
The amount of glucose present in body fluids is about 20 g, and
that readily available from glycogen is approximately 190 g.
During period of fasting glycogen in liver is mobilized but it only
lasts 12 to 24 hours and this source of glucose may not fulfill
metabolic need.
During a longer period of starvation organism must synthesize
glucose from smaller noncarbohydrate precursor molecules.
Gluconeogenesis – synthesis of glucose from
noncarbohydrate precursors
• Liver and kidney are major sites of glucose synthesis
• Main precursors: lactate, pyruvate, glycerol and some
amino acids
• Under fasting conditions, gluconeogenesis supplies
almost all of the body’s glucose
• Gluconeogenesis – universal pathway. It present in
animals, microorganisms, plants and fungi
• Plants synthesize glucose from CO2 using the energy of
sun, microorganisms – from acetate and propionate
Gluconeogenesis is not a Reversal Glycolysis
In glycolysis, glucose is converted into pyruvate; in
gluconeogenesis, pyruvate is converted into glucose.
However, gluconeogenesis is not a reversal of glycolysis.
There are three irreversible reactions in glycolysis catalyzed
by hexokinase, phosphofructokinase, and pyruvate kinase.
1. Glucose + ATP  glucose-6-phosphate + ADP
kcal mol-1
(hexokinase)
G = -8
3. Fructose-6-phosphate + ATP  fructose-1,6-biphosphate + ADP
(phosphofructokinase) G = -5.3 kcal mol-1
10. Phosphoenolpyruvate + ADP  pyruvate + ATP (pyruvate kinase) G = -4
kcal mol-1
These three reactions must be bypassed in gluconeogenesis
Bypassed Reactions in Gluconeogenesis
1. Phosphoenolpyruvate is formed from pyruvate by way of
oxaloacetate through the action of pyruvate carboxylase and
phosphoenolpyruvate carboxykinase.
Pyruvate + CO2 + ATP + H2O  oxaloacetate + ADP + Pi + 2H+
Oxaloacetate + GTP  phosphoenolpyruvate + GDP + CO2
2. Fructose 6-phosphate is formed from fructose 1,6bisphosphate.
Enzyme - fructose 1,6-bisphosphatase.
Fructose 1,6-bisphosphate + H2O  fructose 6-phosphate + Pi
3. Glucose is formed by hydrolysis of glucose 6-phosphate in a
reaction catalyzed by glucose 6-phosphatase.
Glucose 6-phosphate + H2O  glucose + Pi
Gluconeogenesis
The distinctive
reactions are shown in
red.
Comparison of
glycolysis and
gluconeogenesis
Bypass I: Pyruvate  Phosphoenolpyruvate
The first step in gluconeogenesis is the carboxylation of pyruvate
to form oxaloacetate at the expense of a molecule of ATP.
Enzyme pyruvate carboxylase is present only in mitochondria.
Pyruvate is transported into mitochondria from cytoplasm; the
part of pyruvate is formed in mitochondria from amino acids.
Essential cofactor of pyruvate carboxylase is biotin, which
serves as a carrier of CO2.
Biotin-binding domain of
pyruvate carboxylase
Structure of carboxybiotin
Oxaloacetate is polar molecule and can
not pass through the mitochondria
membrane into cytoplasm
Therefore it is reduced:
oxaloacetate + NADH2  malate + NAD+
Enzyme – malate dehydrogenase
Malate passes through the mitochondria
membrane into cytoplasm and again oxidized
to oxaloacetate (enzyme malate
dehydrogenase):
malate + NAD+  oxaloacetate + NADH2
Cytoplasmic oxaloacetate is
decarboxylated to phosphoenolpyruvate
by phosphoenolpyruvate carboxykinase
Phosphoenolpyruvate carboxykinase reaction
Reaction takes place in the cytosol.
In decarboxylation reaction GTP donates a phosphoryl group.
Oxaloacetate is simultaneously decarboxylated and
phosphorylated by phosphoenolpyruvate carboxykinase.
One molecule of ATP and one molecule of GTP were spent to
lift pyruvate to the energy level of phosphoenlpyruvate.
Mechanism of phosphoenolpyruvate carboxykinase reaction
Bypass II: Fructose 1,6-bisphosphate 
Fructose 6-phosphate
• A metabolically irreversible reaction
• The enzyme responsible for this step is fructose
1,6-bisphosphatase
• F1,6BPase is allosterically inhibited by AMP and
fructose 2,6-bisphosphate (F2,6BP)
Bypass III: Glucose 6-phosphate  glucose
In most tissues, gluconeogenesis ends with the formation of
glucose 6-phosphate (G-6P).
Glucose 6-phosphate, unlike free glucose, cannot diffuse
out of the cell.
The generation of free glucose is controlled in two ways:
enzyme responsible for the conversion of glucose 6-phosphate into glucose, glucose 6-phosphatase, is regulated;
enzyme is present only in tissues whose metabolic duty is
to maintain blood-glucose homeostasis — liver and to a
lesser extent kidney, pancreas, small intestine.
Pyruvate carboxylase is allosterically activated by acetyl CoA.
Accumulation of acetyl CoA from fatty acid oxidation
signals abundant energy, and directs pyruvate to
oxaloacetate for gluconeogenesis.
Pyruvate carboxylase reaction
biotin
This reaction takes place in mitochondria matrix.
The final step in the generation of glucose does not take
place in the cytosol.
G-6-P is transported into the lumen of the endoplasmic
reticulum, where it is hydrolyzed by glucose 6phosphatase, which is bound to the membrane.
Glucose 6-phosphatase reaction
Ca2+-binding stabilizing protein is essential for
phosphatase activity.
Glucose and Pi are then shuttled back to the cytosol by
transporters.
Generation of glucose from glucose 6-phosphate. Several proteins play a role
in the generation of glucose. T1 transports G-6-P into the lumen of the ER; T2
and T3 transport Pi and glucose respectively back into the cytosol. SP – Cabinding protein.
The Net Reaction of Gluconeogenesis
2 Pyruvate + 2 NADH + 4 ATP + 2 GTP + 6 H2O 
Glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi + 2H+
G°' = -9 kcal mol-1
Six nucleotide triphosphate molecules are hydrolyzed
to synthesize glucose from pyruvate in gluconeogenesis,
whereas only two molecules of ATP are generated in
glycolysis in the conversion of glucose into pyruvate.
The extra cost of gluconeogenesis is four high
phosphoryl-transfer potential molecules per molecule of
glucose synthesized from pyruvate.
Subcellular Locations of
Gluconeogenic Enzymes
• Gluconeogenesis enzymes are cytosolic
except:
(1) Glucose 6-phosphatase (endoplasmic
reticulum)
(2) Pyruvate carboxylase (mitochondria)
(3) Phosphoenolpyruvate carboxykinase
(cytosol and/or mitochondria)
Regulation of Gluconeogenesis
Gluconeogenesis and glycolysis are reciprocally regulated
- within a cell one pathway is relatively inactive while the
other is highly active.
The amounts and activities of the distinctive enzymes of
each pathway are controlled.
The rate of glycolysis is determined by the concentration
of glucose.
The rate of gluconeogenesis is determined by the
concentrations of precursors of glucose.
AMP stimulates phosphofructokinase, whereas ATP and
citrate inhibit it. Fructose 1,6bisphosphatase is inhibited by
AMP and activated by citrate.
Fructose 2,6-bisphosphate
strongly stimulates phosphofructokinase 1 and inhibits
fructose 1,6-bisphosphatase.
During starvation, gluconeogenesis predominates because
the level of F-2,6-BP is very low.
High levels of ATP and alanine,
which signal that the energy
charge is high and that building
blocks are abundant, inhibit the
pyruvate kinase.
ADP inhibits phosphoenol-pyruvate carboxykinase.
Pyruvate carboxylase is
activated by acetyl CoA and Gluconeogenesis is favored when the cell is rich
inhibited by ADP.
in biosynthetic precursors and ATP.
Regulation of the Enzymes Amount by Hormones
Hormones affect gene expression primarily by changing the
rate of transcription.
Insulin, which rises subsequent to eating, stimulates the
expression of phosphofructokinase and pyruvate kinase.
Glucagon, which rises during starvation, inhibits the
expression of these enzymes and stimulates the production
of phosphoenolpyruvate carboxykinase and fructose 1,6bisphosphatase.
Transcriptional control in eukaryotes is much slower than
allosteric control; it takes hours or days in contrast with
seconds to minutes.
Precursors for Gluconeogenesis
• Any metabolite that can be converted to
pyruvate or oxaloacetate can be a glucose
precursor
• Major gluconeogenic precursors in mammals:
(1) Lactate
(2) Most amino acids (especially alanine),
(3) Glycerol (from triacylglycerol hydrolysis)