Transcript Lecture 33 - University of Arizona

Carbohydrate Metabolism 1:
Pentose Phosphate Pathway,
Gluconeogenesis, Reciprocal Regulation
Bioc 460 Spring 2008 - Lecture 33 (Miesfeld)
primaquine
Deficiencies in the enzyme
glucose-6P dehydrogenase
affects 400 million people
The dual function enzyme
PFK-2/FBPase-2 controls flux
through gluconeogenesis and
glycolysis by controlling levels
of F-2,6-BP in the cell
Athletes like Jenna Gresdal
rely on the Cori Cycle to
maintain glucose levels
Key Concepts: The Pentose Phosphate Pathway
• The pentose phosphate pathway takes place entirely within the
cytoplasm and is also known as the hexose monophosphate shunt or
phosphogluconate pathway.
• The most important function of the pentose phosphate pathway is to
reduce two molecules of NADP+ to NADPH (nicotinamide adenine
dinucleotide phosphate) for each glucose-6-phosphate that is oxidatively
decarboxylated to ribulose-5-phosphate.
• NADPH is functionally similar to NAD+ however, NADPH is the primary
reductant in the cell, whereas, NAD+ is the primer oxidant. NADPH is
critical to maintaining reduced glutathione levels in cells which is
required to minimized damage from reactive oxygen species.
• The pentose phosphate pathway is also responsible for producing
ribose-5-phosphate which provides the ribose sugar backbone that
anchors the nucleotide base to DNA and RNA polymers.
We will cover three primary
pathways related to
carbohydrate metabolism in
non-photosynthetic organisms:
1. Pentose phosphate pathway
2. Gluconeogenesis
3. Glycogen metabolism
Metabolism of ribose sugars in
the pentose phosphate
pathway is used to generate
NADPH and to provide the
carbohydrate component of
nucleotides.
The major sources of carbon in
gluconeogenesis are amino
acids and glycerol in animals,
and glyceraldehyde-3phosphate (GAP) in plants.
Pathway Questions
1. What does the pentose phosphate pathway accomplish for the cell?
– The oxidative phase generates NADPH which is required for
many biosynthetic pathways and for detoxification of reactive
oxygen species.
– The nonoxidative phase interconverts C3, C4, C5, C6 and C7
monosaccharides to produce ribose-5P for nucleotide synthesis,
and also to regenerate glucose-6P to maintain NADPH
production by the oxidative phase.
Pathway Questions
2. What is the overall net reaction of the pentose phosphate pathway
when it is utilized to generate the maximum amount of NADPH?
6 Glucose-6P + 12 NADP+ + 12 H2O →
5 Glucose-6P + 12 NADPH + 12 H+ + 6 CO2
Pathway Questions
3. What are the key enzymes in the pentose phosphate pathway?
Glucose-6P dehydrogenase (G6PD)–This reaction is the commitment
step in the pathway and is feedback-inhibited by NADPH.
Transketolase and Transaldolase - together these two enzyme
catalyze the reversible "carbon shuffle" reactions of the
nonoxidative phase of the pathway.
Pathway Questions
4. What are examples of the pentose phosphate pathway in real life?
Glucose-6P dehydrogenase G6PD) deficiency is the most common
enzyme deficiency in the world and affects over 400 million
people. Red blood cells do not produce enough NADPH to protect
against reactive oxygen species that are generated by anti-malarial
drugs (primaquine) and by compounds in fava beans (vicine).
Malaria-infected Anopheles
mosquito biting a human
A big bowl of fava beans
Two Phases of the
Pentose Phosphate
Pathway
The pentose
phosphate
pathway can be
divided into two
phases, the
oxidative phase,
which generates
NADPH, and the
nonoxidative
phase, which
regenerates G-6P.
Three enzymatic reactions in the oxidative phase
1
2
3
G6PD is the commitment step in the Pentose Phosphate
Pathway because 6-Phosphogluconon-d-lactone has no other
metabolic fate except to be converted to 6-phosphogluconate.
1
2
3
4
The non-oxidative
phase of the PPP
The carbon shuffle
reactions of the
nonoxidative phase are
used to regenerate
glucose-6P using the
same transketolase and
transaldolase enzyme
reactions as the Calvin
Cycle.
6
5
Six glucose-6P (36
carbons) are metabolized
to regenerate five glucose6P (30 carbons).
What happened to 6 carbons?
1
2
3
4
5
Metabolic flux
through the Pentose
Phosphate Pathway
is tightly-regulated
• If increased NADPH
is required for
biosynthetic pathways.
• If cells need to
replenish nucleotide
pools.
• If ATP levels in the
cell are low.
Regulation of the G6PD activity controls flux through the
glycolytic pathway and pentose phosphate pathways
Glucose-6P dehydrogenase deficiency in humans
The pentose phosphate pathway is responsible for maintaining high
levels of NADPH in red blood cells (erythrocytes) for use as a
reductant in the glutathione reductase reaction. Glutathione is a
tripeptide that has a free sulfhydryl group which functions as an
electron donor in a variety of coupled redox reactions in the cell.
Glucose-6P dehydrogenase deficiency in humans
When erythrocytes are
exposed to chemicals
that generate high levels
of superoxide radicals,
GSH is required to
reduce these damaging
compounds.
The pentose phosphate
pathway in erythrocytes
normally provides
sufficient levels of
NADPH to maintain the
GSH:GSSG ratio at
about 500:1.
Glucose-6P dehydrogenase deficiency in humans
Primaquine inhibits growth of the malarial parasite in red blood cells by creating a
hostile environment. The biochemical basis for this drug-induced illness was
found to be lower than normal levels of NADPH due to a G6PD deficiency.
The acute hemolytic anemia seen in individuals with G6PD who are treated with
primaquine explains the symptoms of favism. One of the compounds in fava
beans is vicine, a toxic glycoside that induces oxidative stress in erythrocytes.
What might explain the observation that cultures with high amounts of fava
beans in the diet were associated (in ancient times) with low malaria rates?
Key Concepts: Gluconeogenesis
• The importance of gluconeogenesis is to provide glucose for cells from
non-carbohydrate precursors.
• Three steps in glycolysis must be bypassed by gluconeogenic enzymes
in order to overcome large G differences.
• Reciprocal regulation at the PFK-1 (glycolysis) and F-1,6-BPase
(gluconeogenesis) is controlled by the allosteric regulator F-2,6bisphosphate, as well as, energy charge (ATP/AMP), and citrate levels.
• The Cori Cycle recycles lactate produced in anaerobic muscle cells
during exercise by exporting it to the liver where it is converted to
pyruvate and used to synthesize glucose by gluconeogenesis.
Pathway Questions
1. What does gluconeogenesis
accomplish for the organism?
The liver and kidney generate
glucose from noncarbohydrate
sources.
Plants use the gluconeogenic
pathway to convert GAP into
glucose which is used to make
sucrose and starch.
Pathway Questions
2. What is the overall net reaction of gluconeogenesis?
2 pyruvate + 2NADH + 4ATP + 2GTP + 6H2O →
Glucose + 2NAD+ + 2H+ + 4ADP + 2GDP + 6Pi
3. What are the key enzymes in gluconeogenesis?
Pyruvate carboxylase converts pyruvate to oxaloacetate.
Phosphoenolpyruvate carboxykinase (PEPCK) converts oxaloacetate
to phosphoenolpyruvate (PEP).
Fructose-1,6-bisphosphatase-1 (FBPase-1) catalyzes the
dephosphorylation of fructose-1,6BP to form fructose-6P.
Glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6P
to form glucose.
4.Application of
gluconeogenesis
in real life
Pathway Questions
Glucose monitoring devices
are based on an assay using
the enzyme glucose oxidase
which produces gluconate
and hydrogen peroxide
(H2O2) from glucose.
The level of H2O2 in the
sample is detected by an
indicator dye that is oxidized
in a reaction catalyzed by
peroxidase.
Gluconeogenesis and glycolysis
are opposing pathways
Two of the bypass enzymes in
gluconeogenesis, fructose1,6-bisphosphatase-1
(FBPase-1) and glucose-6phosphatase, simply reverse
the reaction
However, 4 extra ATP/GTP,
and pyruvate carboxylase and
phosphoenolypyruvate
carboxykinase (PEPCK), are
required to catalyze the
bypass reaction that converts
pyruvate to PEP.
How do you come up with the
4 extra ATP/GTP for
gluconeogenesis compared
to glycolysis?
Reciprocal regulation of PFK-1 and FBPase-1
The activities of PFK-1 and FBPase-1 are regulated by the allosteric
effectors AMP, citrate, and fructose-2,6-bisphosphate (F-2,6-BP), but in a
reciprocal manner.
What is the metabolic logic of reciprocal regulation by citrate?
Reciprocal regulation of PFK-1 and FBPase-1
In the presence of F-2,6-BP,
the affinity of PFK-1 for its
substrate fructose-6P is 25
times higher than it is in the
absence of F2,6BP.
Looking at the activity curves
for FBPase-1 in the presence
and absence of F-2,6-BP it
can be seen that the affinity of
FBPase-1 for its substrate
fructose-1,6BP is 15 times
lower in the presence of F2,6-BP.
Levels of F-2,6-BP in the cell are controlled by a dual
function enzyme called PFK-2/FBPase-2
The amount of F-2,6-BP in the cell is controlled by the activity of a dual function
enzyme containing two catalytic activities, 1) a kinase activity called
phosphofructokinase-2 (PFK-2) that phosphorylates fructose-6P to form F-2,6-BP,
and 2) a phosphatase activity called fructose-2,6-bisphosphatase (FBPase-2) that
dephosphorylates F-2,6-BP to form fructose-6P.
Levels of F-2,6-BP in the cell are controlled by a dual
function enzyme called PFK-2/FBPase-2
When the PFK-2/FBPase-2 dual function enzyme is unphosphorylated, then the
PFK-2 activity in the enzyme is stimulated resulting in the net phosphorylation of
fructose-6P to produce more F-2,6-BP which stimulates glycolytic flux.
In contrast, when PFK-2/FBPase-2 is phosphorylated, the activity of FBPase-2
is stimulated, leading to less F-2,6-BP, and reduced flux through glycolysis,
with a concomitant increase in flux through gluconeogenesis.
Less F-2,6-BP
and gluconeogenesis
More F-2,6-BP
and glycolysis
Levels of F-2,6-BP in the cell are controlled by a dual
function enzyme called PFK-2/FBPase-2
Activation of the glucagon receptor in liver cells results in stimulation of protein
kinase A signaling, leading to an increase in gluconeogenesis, whereas, insulin
signaling stimulates protein phosphatase-1, leading to an increase in glycolysis
.
The Cori Cycle
The Cori cycle provides a mechanism to convert lactate produced by anaerobic
glycolysis in muscle cells to glucose using the gluconeogenic pathway in liver
cells. The Cori cycle has an energy cost (4 ATP equivalents), but it is worth it.
These 4 extra ATP/GTP come from the fact that glycolysis yields a net of 2 ATP
per glucose, but gluconeogenesis costs a total of 4 ATP and 2 GTP per glucose.
The Cori Cycle is important
for peak performance in
athletic competition
Athletes "warm down" after exercise to
enhance circulation so that lactate will be
cleared from the muscle and be used in the
liver for glucose synthesis via the Cori cycle.