Gluconeogenesis
NT Mazomba
2022
19 February 2022
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Objectives
1.
2.
3.
4.
What, where, when and why?
Relationship with glycolysis Bypasses and transport of OAA
Regulation
Synthesis of glycogen – why and
how?
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Introduction
Pathway for biosynthesis of new glucose
from noncarbohydrate precursors (i.e.
not from stored glycogen) –
•
Occurs in plants, microorganisms and
animals
•
Occurs mainly in the liver (ca. 90%) and
lesser in kidney cortex (ca. 10%) - in
mitochondria and cytoplasm
•
When dietary glucose is not available,
liver glycogen supplies are depleted. This
occurs during heavy/intense exercise,
diabetes, fasting or prolonged
starvation.
Why?
1. Glucose is essential as a primary source
of energy for the brain, testes,
erythrocytes and kidney medulla
2. It provides precursors for glycogen
storage in other tissues, such as liver and
muscles
Precursors
•
Glycolysis products (pyr, lactate, DHAP),
TCA intermediates (OAA, α-kg), and
carbon skeletons of glucogenic (?)
19 February
2022(Ala, Asp, Asn, Glu, Gln)
amino
acids
•
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3
Glycolysis/Gluconeogenesis
•
The following
discussion will focus on
gluconeogenesis that
occurs in higher
animals, and in
particular in the liver of
mammals.
•
Synthesis of glucose
from three and four
carbon precursors - a
reversal of glycolysis?
•
What is the difference?
Look closely…. start
with glucose or
pyruvate.... Are there
any similarities or
differences?
•
Irreversible enzymatic
steps of glycolysis are
bypassed using
different reactions or
enzyme(s) in
gluconeogenesis
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Bypass 1 - Pyr to PEP
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Conversion of PYR to PEP in gluconeogenesis requires two steps
This is the reverse of an exergonic reaction in glycolysis, PEP to PYR
1st step - ATP-requiring reaction catalyzed by pyruvate carboxylase (PC)
Firstly, pyruvate is carboxylated to form oxaloacetate (OAA)
The CO2 used in this reaction is in the form of bicarbonate (HCO3-)
An anaplerotic reaction - can be used to fill-up the TCA cycle
Needs Mg2+ and biotin
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OAA transport
• For gluconeogenesis to proceed, the OAA produced
by PC must be transported to the cytosol
• No transport mechanism exist for OAA’s direct transfer
and OAA will not diffuse freely through the mitochondrial
membrane
• Mitochondrial OAA can become cytosolic only via
three ways;
1. Conversion to PEP by the mitochondrial phosphoenol carboxykinase
(PEPCK),
2. Transamination to aspartate or,
3. Reduction to malate
• All of these (i.e. PEP, aspartate, malate) are
transported to the cytosol
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OAA decarboxylated to PEP
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If OAA is converted to PEP by mitochondrial PEP carboxykinase
(PEPCK), it is transported to the cytosol where it is a direct substrate for
gluconeogenesis and nothing further is required
PEPCK requires GTP for the decarboxylation of OAA to yield PEP
No net fixation of carbon occurs – PYR and PEP have each 3 carbons
Already 2 ATPs have been used in the conversion of PYR to PEP
All of these steps occur within the mitochondrion
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Transamination of OAA to aspartate
• Aspartate is transported to the cytosol where the
reverse transamination occurs yielding cytosolic
OAA
• This transamination reaction requires continuous transport of glutamate into,
and -ketoglutarate out of the mitochondrion
• Therefore, this process is limited by the availability of these substrates
• Whether mitochondrial decarboxylation or
transamination occurs, is a function of the
availability of PEPCK or transamination
intermediates
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OAA reduced to malate
• Mitochondrial OAA - reduced to malate in a reversal of the TCA
cycle reaction catalyzed by malate dehydrogenase (MDH)
• The reduction of OAA to malate requires NADH
• The resultant malate is transported to the cytosol where it is
oxidized to OAA by cytosolic MDH which requires NAD+ and
yields NADH
• When in the cytoplasm, OAA is converted to PEP by the
cytosolic version of PEPCK
• Hormonal signals control the level of PEPCK protein as a
means to regulate the flux through gluconeogenesis
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Bypass 2 - F-1,6-bP to F-6-P
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Fructose-1,6-bisphosphate (F1,6BP) - simple hydrolysis - catalyzed
by fructose-1,6-bisphosphatase (F1,6BPase) - to fructose-6phosphate (F6P) is the reverse of the rate limiting step of
glycolysis.
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Major point of control of gluconeogenesis.
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What does this remind you of in glycolysis?
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Bypass 3 - G-6-P to Glc (or Glycogen)
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Simple hydrolysis - G6P to glucose (glucose-6phosphatase, G6Pase)
Similar to F1,6BPase reaction
Most non-hepatic tissues, the brain and skeletal muscle,
lack G6Pase activity
In the kidney, muscle and especially the liver, G6P can be
shunted toward glycogen synthesis if blood glucose levels
are adequate.
2007 BCH311 Gluconoegenesis NTM
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Regulation of gluconeogenesis
• Gluconeogenesis and glycolysis both occur mainly in the cytosol
• Gluconeogenesis synthesizes glucose and glycolysis catabolizes
glucose
• The two pathways must be controlled in a reciprocal fashion, i.e.
negative effectors of glycolysis must be positive effectors of
gluconeogenesis and vice-versa
• The interconversion of fructose-6-phosphate and fructose-1,6bisphosphate is the key control point in both gluconeogenesis and
glycolysis
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• Regulation
of the two
pathways
is
primarily
brought
about by
allosteric
controls
on the
enzymes
that differ
between
the two
pathways
• These
enzymes
are:
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Regulation of gluconeogenesis
• The conversion of fructose-1,6bisphosphate and fructose-6phosphate is the key control point
in gluconeogenesis
• The major allosteric regulatory
factor of the two pathways is
fructose-2,6-bisphosphate.
• Phosphofructokinase 2 (PFK 2)
and Fructose-2,6-bisphosphatase
(F2,6BPase) are enzymes that are
involved in the biosynthesis
(kinase) and degradation
(phosphatase) of Fructose-2,6bisphosphate respectively.
• These functions occur on the same
peptide, a bifunctional regulatory
enzyme and are affected differently
by phosphorylation
• Interconversion of PFK 2 and
F2,6BPase depends on the level of
cAMP (which is stimulated by
glucagon and is inhibited by
insulin)
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Regulation of gluconeogenesis
• In the absence of glucose
(e.g. during starvation), the
hormones glucagon is
released by the pancreas and
attach to cells (e.g. liver) that
carry out gluconeogenesis
• Their attachment trigger a
cascade of events inside the
cells
• These include increased
levels or high levels of cAMP
which stimulates
phosphorylation of the
bifunctional peptide, favoring
the F2,6BPase function.
• This enzyme degrades F2,6BP
to F6P.
• The low levels or absence of
F2,6BP lead to activation of
F1,6BPase, which in turn
catalyze the conversion of
F1,6BP to F6P.
• This sequence of events thus
increase the rate of
gluconeogenesis.
• What happens when there are
high levels of glucose?
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Regulation of gluconeogenesis
• What happens when there
are high levels of glucose,
i.e. in a fed state?
• Insulin is released in order to
promote the uptake of
glucose by the muscles and
adipose tissue.
• This again leads to a
cascade of events
• These include lowered
levels of cAMP which
stimulates
dephosphorylation of the
bifunctional peptide,
favoring the PFK2 function.
• This enzyme catalyzes the
synthesis of F2,6BP from
F6P.
• The high levels of F2,6BP
lead to inhibition of
F1,6BPase and activation of
PFK1, which catalyzes the
conversion of F6P to F1,6BP.
• This sequence of events thus
increase the rate of
glycolysis.
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Glycogen synthesis
• A major fate of abudant
glucose in animals is the
synthesis of glycogen
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Glycogen synthesis
• A major fate of abudant
glucose in animals is the
synthesis of glycogen
• 1st reaction - hexokinase,
glc to glc-6-P
• 2nd reaction phosphoglucomutase interconversion of glc-6-P
and glc-1-P
• This enzyme serves as a
common link between
glycogen biosynthesis
and glycogen breakdown
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Glycogen synthesis
• 3rd reaction – UDPglucose
pyrophosphorylase - the
transfer of glucose (from
glc-1-P) to uridine
triphosphate, forming
UDP-glucose and
pyrophosphate
(“activating”)
• This reaction is made
essentially irreversible by
hydrolysis of the
pyrophosphate to 2Pi.
• Effect of this is that
glucose is prepared for
incorporation into the
growing glycogen chain
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Glycogen synthesis
• This enzyme cannot simply form a bond between two
isolated glucose molecules; it must add to an existing chain
with α(1,4) glycosidic linkages.
• The initiation of glycogen synthesis requires a primer for
this reason. The hydroxyl group of a specific tyrosine of the
protein glycogenin (37,300 Da) serves this purpose.
• In the first stage of glycogen synthesis, a glucose residue is
linked to this tyrosine hydroxyl, and glucose residues are
successively added to this first one.
• The glycogenin molecule itself acts as the catalyst for
addition of glucoses until there are about eight of them
linked together. At that point, glycogen synthase takes over.
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Glycogen synthesis
• 4th reaction glycogen synthase - the formation of an α(1-4) glycosidic
bond between carbon 4 of an existing glycogen chain and carbon 1 of a
glucose (from UDP-glucose).
• UDP is released in the process.
• Glycogen
synthase is the major point of regulation of glycogen
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biosynthesis.
Glycogen synthesis
• 5th reaction amylo-(1,4->1,6)-transglycosylase - most commonly called
branching enzyme.
• Introduces the abundant α(1-6) branches, which are characteristic of glycogen
molecules.
• The reaction transfers a terminal fragment, some 6 or 7 residues long, from a
branch terminus at least 11 residues in length to a hydroxyl group at the 6position of a glucose residue in the interior of the polymer
• As a result of the transfer, two termini recognized by glycogen synthase are
created where only one existed before.
• The synthesis and the mobilization of glycogen can proceed quickly and
efficiently, depending on the needs of the cell.
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Synthesis of other complex sugars
• Involves many of the similar mechanisms as for glycogen, particularly the
use of nucleotide-linked sugars as activated biosynthetic intermediates
and glycosyltransferase enzymes.
Cellulose - a glucose homopolymer with β(1-4) linkages between the
units.
• UDP-glucose is used as an intermediate in some plant species.
• Others use ADP-glucose and CDP-glucose.
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