GLUCONEOGENESIS
Student Edition
5/30/13 Version
Dr. Brad Chazotte
213 Maddox Hall
chazotte@campbell.edu
Web Site:
http://www.campbell.edu/faculty/chazotte
Original material only ©2000-14 B. Chazotte
Pharm. 304
Biochemistry
Fall 2014
Goals
•Learn the enzymes and step of the gluconeogenesis pathway.
•Learn the similarities and difference the between gluconeogenesis and glycolysis
pathways.
•Understand the principles for biosynthetic (anabolic) pathways vs catabolic.
•Understand how the gluconeogenesis pathway is regulated and how it is regulated vs
glycolysis.
•Understand the concept and benefits of metabolic burden sharing
•Be aware the Cori cycle.
•Understand how substrate cycles can amplify metabolic signals and/or produce heat.
Biosynthetic (Anabolic) Pathways
•Use chemical energy (ATP, NADH, NADPH) to synthesize
cellular components from simple precursor molecules.
•Generally reductive rather than oxidative.
•Anabolism and catabolism proceed simultaneously in a
dynamic steady-state to maintain the “intricate orderliness of
living cells”.
Lehninger 2000 p722
Organizing Principles of Biosynthesis 1
1.
Molecules are synthesized and degraded by different pathways.
a. Two opposing anabolic and catabolic pathways may share many
reversible reactions.
b. Each pathway has at least one unique and essentially irreversible
reaction.
c. This insures that the carbon flow through these pathways is dictated
by the cell’s changing requirements for energy, precursors, and
macromolecules rather than simple mass action.
Lehninger 2000 p722
Organizing Principles of Biosynthesis 2
2.
Corresponding anabolic and catabolic pathways are controlled by
one or more reactions unique to each pathway.
a. Opposing pathways are regulated in a coordinated, reciprocal manner
so that the stimulation of the anabolic pathway is accompanied by the
inhibition of the catabolic one and vice versa.
b. Biosynthetic pathways are typically regulated at an early, exergonic
step that commits intermediates to the pathway and does not waste
energy by making unneeded intermediates.
Lehninger 2000 p722
Organizing Principles of Biosynthesis 3
3. Energy requiring biosynthetic processes are coupled to the breakdown of
ATP in such a way that the overall process is essentially irreversible in
vivo.
a. Thus the total amount of energy from ATP and NAD(P)H used
always exceeds the minimum free energy needed to convert the
precursor into the biosynthetic product.
b. Consequently the biosynthetic process is thermodynamically
favorable (G < 0) even for low precursor concentrations.
Lehninger 2000 p722
Carbohydrate Biosyntheses from Simple Precursors
The pathway from PEP to glucose-6-P
is common to the biosynthetic
conversion of many different
precursors in animals and plants.
Gluconeogenesis, meaning
“formation of new sugar”, occurs in
all animals, plants, fungi, and
microorganisms. In most cases the
reactions are essentially the same.
Gluconeogenesis
Lehninger 2000 Fig 20.1
Importance of Gluconeogenesis
The brain depends on glucose as its primary fuel using ~120g/day out
of ~160g/day for the typical adult human.
Red blood cells use only glucose as a fuel.
Only 20g are present in the body fluids and ~190 g are available from
glycogen storage.
Therefore, total reserves of glucose are about a single day’s supply.
For longer periods of starvation, glucose must be formed from
noncarbohydrate sources.
Pyruvate
Noncarbohydrate Precursors for
Gluconeogenesis
Any metabolite that can be converted to pyruvate or oxaloacetate can be a
glucose precursor
Lactate is formed by active skeletal muscle when glycolysis > oxidative
metabolism. Lactate is converted by lactate dehydrogenase to pyruvate.
Pyruvate
(Certain) Amino acids are derived from protein in the diet or from muscle
protein catabolism during starvation. Carbon skeletons of most amino acids
are catabolized to pyruvate or citric acid cycle intermediates
Glycerol is the result of a breakdown of triacylglycerols in fat cells. Fatty
acids also result, but cannot be used by animals to make glucose. Glycerol
enters glycolysis or gluconeogenesis at dihydroxyacetone phosphate
Glycerol’s Entry in Gluconeogenesis or
Glycolysis
Gluconeogenesis
Berg, Tymoczko & Stryer, 2012 Chap. 16 np.481
The Gluconeogenesis Balanced
Equation
2Pyruvate + 2NADH +4H+ + 4ATP +2GTP +6H2O
Glucose +2NAD+ + 4ADP + 2GDP + 6Pi
Gº = –16 kJ/ mol
Because of the presence of separate gluconeogenic enzymes at the three irreversible steps in the
glycolytic pathway that converts glucose to pyruvate, glycolysis and gluconeogenesis both are
rendered THERMODYNAMICALLY favorable.
Gluconeogenesis
Voet & Voet 1995 Chap 21 P.604
Gluconeogenesis Pathway
Gluconeogenesis
Berg, Tymoczko & Stryer, 2012 Fig. 16.24
Voet, Voet & Pratt 2013 Figure 16.15
Subcellular Location of Gluconeogenic
Enzymes
• Gluconeogenesis enzymes are cytosolic except:
(1) Glucose 6-phosphatase (endoplasmic reticulum)
(2) Pyruvate carboxylase (mitochondria)
(3) PEPCK (cytosol and/or mitochondria)
Mitochondrial pyruvate can:
1) be converted to citrate and used in the cytosol for
the synthesis of fatty acids.
2) be changed to acetyl CoA and enter the citric acid
cycle.
3) be converted by pyruvate carboxylase to
oxaloacetate for gluconeogenesis.
Horton et al., 2000 Chap 13.6
G’s of Erythrocyte Glycolytic Rxs
Lehninger 2000 Table 20.1
Sequential Rx in Gluconeogenesis
from Pyruvate
Lehninger 2000 Table 20.2
Gluconeogenesis Pathway I
Gluconeogenesis
Berg, Tymoczko & Stryer, 2012 Fig. 16.24b
Conversion of Pyruvate into Phosphoenolpyruvate
Pyruvate carboxylase
Pyruvate + CO2 + ATP + H2O
oxaloacetate + ADP + Pi + 2H+
•The bypass of glycolysis’ pyruvate kinase reaction requires two
separate reactions in gluconeogenesis.
•One of the anaplerotic reactions that is used to maintain the
levels of intermediates in the citric acid cycle
Gluconeogenesis
G’ = -2.1 kJ mol -1
Voet, Voet & Pratt 2013 Fig 16.16
Synthesis or PEP from Pyruvate I
Gluconeogenesis
Lehninger 2000 Fig 203a
Pyruvate Carboxylase: Rx Mechanism
Pyruvate Carboxylase catalyzes the ATPdriven synthesis of oxaloacetate from
pyruvate and HCO3-. This reaction occurs in
two phases.
Phase I is a three step reaction sequence.
Biotin is carboxylated at its N1’ position by a
bicarbonate ion.
Phase II in this phase the activated carboxyl
group is transferred to pyruvate from
carboxybiotin in a three step reaction sequence
to form oxaloacetate.
Gluconeogenesis
Voet , Voet & Pratt 2008 Fig 16.18
Domain Structure of Pyruvate Carboxylase
The ATP grasp domain activates HCO3- and transfers CO2 to the biotin-binding domain.
From there the CO2 is transferred to pyruvate generated in the central domain.
Gluconeogenesis
Berg, Tymoczko & Stryer, 2012 Fig. 16.25
Pyruvate Carboxylase’s Biotin-binding Domain
Key feature: biotin is on a flexible tether, attached to the -amino group of lysine, allowing it
to move between the ATP-bicarbonate site and the pyruvate site.
Gluconeogenesis
Berg, Tymoczko & Stryer, 2012 Fig. 16.26
Carboxybiotin
Berg, Tymoczko & Stryer, 2012 Fig. 16.27
Gluconeogenesis
Voet, Voet & Pratt 2013 Fig 16.17b
Opposing Pathways of Gluconeogenesis and Glycolysis II
Gluconeogenesis
Lehninger 2000 Fig 20.2b
Conversion of Oxaloacetate into Phosphoenolpyruvate
phosphoenolpyruvate
carboxykinase
Oxaloacetate + GTP
Phosphoenolpyruvate + CO2 + GDP
G’ = 2.9 kJ mol -1
Gluconeogenesis
Synthesis or PEP from Pyruvate II
Oxaloacetate is converted to phosphoenolpyruvate in the cytosol by PEP carboxykinase in a
reaction that requires Mg2+ and GTP as the phosphoryl donor.
Voet, Voet & Pratt 2013 Figure 16.19
Gluconeogenesis
PEP Carboxykinase: Rx Mechanism
A monomeric, 74 kD enzyme that
catalyzes a GTP-driven decarboxylation
of oxaloacetate to form PEP and GDP
The CO2 that carboxylates pyruvate
to synthesize oxaloacetate is
eliminated in the formation of PEP.
Gluconeogenesis
Voet & Voet Biochemistry 1995 Fig. 21.5
The Mitochondrion
Supplies Malate made
from Pyruvate for use in
Gluconeogenesis in the
Cytosol
Gluconeogenesis
Note: the starting molecule is pyruvate.
•Inside the mitochondrion
oxaloacetate is reduced to
malate in order to be
transported outside the
mitochondrion
•Once the malate is
transported outside (via the
malate - -ketoglutarate
shuttle) it is re-oxidized to
oxaloacetate
Berg, Tymoczko & Stryer, 2012 Fig. 16.28
Alternate Paths from Pyruvate to PEP
This oxaloacetate
is converted
directly to PEP by
a mitochondrial
isozyme of PEP
carboxykinase
Pathway predominant
when the starting
molecule is pyruvate
Gluconeogenesis
When lactate is the
precursor this
pyruvate to PEP
bypass is predominant
Lehninger 2000 Fig 20.4
PEP & Oxaloacetate Transport
Cytosol  Mitochondria
PEP has a direct transporter, whereas
oxaloacetate does not and must be converted
for transmembrane passage.
Route 2 involves the conversion of oxaloacetate
to malate AND involves NADH.
Route 1 uses aspartate amino transferase. In
this case oxaloacetate is converted to
aspartate for transport.
PEP is transported between the mitochondrion and
the cell cytosol by a specific membrane transporter
so it can move between the two compartments
depending on the equilibrium.
Voet, Voet & Pratt 2013 Figure 16.20
Sequential Reactions in Gluconeogenesis
(Reprise)
Gluconeogenesis
Lehninger 2000 Table 20.2
Gluconeogenesis Pathway II
Gluconeogenesis
Berg, Tymoczko & Stryer, 2012 Fig. 16.24a
Conversion of Fructose 1,6-bisphosphate
into Fructose -6P and PPi:
An Irreversible Step
Fructose 1,6biphosphatase
Fructose 1,6-bisphosphate + H2O
Gluconeogenesis
fructose 6-phosphate + Pi
G’ = -16.3 kJ mol -1
Conversion of Glucose-6-phosphate into
Glucose: An Irreversible Step
Glucose-6-phosphatase
Glucose-6-phosphate + H2O
Gluconeogenesis
Glucose + Pi
G’ = -12.1 kJ mol -1
Glucose-6-Phosphatase
This enzyme is found primarily in the endoplasmic reticulum of
liver cells. – Why?
Metabolic Control of Glycolysis
and Gluconeogenesis
In cells gluconeogenesis and glycolysis are coordinated such that one is mainly inactive
while the other in highly active. (If both were highly active at the same time the result
would be a FUTILE CYCLE consuming two ATP and two GTP per reaction cycle).
•The rate of glycolysis is typically controlled by the glucose concentration.
•The rate of gluconeogenesis is typically controlled by the concentrations
of lactate and other glucose precursors.
Reciprocal Regulation of Gluconeogenesis and Glycolysis in Liver
IMPORTANT!!
Gluconeogenesis
Berg, Tymoczko & Stryer, 2012 Fig. 16.30
Voet, Voet & Pratt 2013 Fig. 16.21; 16.23
“Subtrate Cycle”
can amplify metabolic signals and produce heat.
Gluconeogenesis
Berg, Tymoczko & Stryer, 2012 Fig. 16.34
Metabolic Burden Sharing
Different organs/tissues in the body can be metabolically linked.
Lactate produced by active skeletal muscle and erythrocytes is an energy source for other
organs.
•Skeletal muscle during vigorous exercise produces pyruvate at a rate faster than
oxidative metabolism via the citric acid cycle can use it.
•Also NADH production is more rapid than its conversion to NAD+ in aerobic
metabolism.
•(Remember that glycolysis needs NAD+ for glyceraldehyde 3-P oxidation to proceed.)
•The cell can oxidize NADH to NAD+ in a reaction that converts pyruvate to lactate via
lactate dehydrogenase. The problem is that lactate can not be further metabolized, only
converted back to pyruvate.
Important: Muscle’s ability to reduce pyruvate to lactate is a way shift the metabolic
burden under high stress to other organs, e.g. liver, heart. (Reaction also regenerates
NAD+ in muscle)
Cori Cycle (an interorgan metabolic “pathway”)
Lactate & pyruvate
diffuse out of active
muscle into blood.
Excess lactate in the
blood enters the liver
where it is converted to
pyruvate and then to
glucose via
gluconeogenesis.
Gluconeogenesis: Cori Cycle
Berg, Tymoczko & Stryer, 2012 Fig. 16.35
Horton et al., 2002 Fig. 13.12
Coordination of Glycolysis and Gluconeogenesis
In liver and kidney glucose is produced by
gluconeogenesis and can go out into the blood to
be used by other tissues such as muscle.
In muscle alanine is formed by a transamination
reaction from pyruvate. Whereas the reverse
process occurs in the liver. This cycling helps to
maintain the nitrogen balance
Muscles and red blood cells can produce lactate
which, as we have seen in the Cori cycle can
travel to the e.g. liver where it is metabolized to
pyruvate, etc
Gluconeogenesis
(Remember: alanine is also a major glucose
precursor)
Berg, Tymoczko & Stryer,
2002 Fig. 16.34
Opposing Pathways of Gluconeogenesis and Glycolysis
I
Gluconeogenesis
Lehninger 2000 Fig 20.2a
End of Lectures