Download Lecture 33

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.
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)– enzyme catalyzing the first
reaction in the pathway which converts glucose-6P to 6phosphogluconolactone. This reaction is the commitment step in
the pathway and is feedback-inhibited by NADPH. Defects in
glucose-6P dehydrogenase cause a dietary condition called favism.
Transketolase and Transaldolase - together these two enzyme
catalyze the reversible "carbon shuffle" reactions of the
nonoxidative phase of the pathway. These are the same enzymes
used in the Calvin Cycle to regenerate ribulose-5P from
glyceraldehyde-3P.
Pathway Questions
4. What are examples of the pentose phosphate pathway in real life?
Glucose-6P dehydrogenase deficiency is the most common enzyme
deficiency in the world and affects over 400 million people. A 90%
decrease in enzyme activity results in the inability of red blood cells to
produce enough NADPH to protect the cells from 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
interconverts C3, C4,
C5, C6 and C7 sugar
phosphates using
many of the same
"carbon shuffle"
reactions we saw in
the Calvin cycle.
Three enzymatic reactions in the oxidative phase
1. Oxidation of glucose-6P by the enzyme glucose-6P
dehydrogenase (G6PD) to 6-phosphogluconolactone is coupled to
the reduction of NADP+ resulting in the formation of one molecule of
NADPH. This is the commitment step in the pathway.
2. 6-phosphogluconolactone is hydrolyzed by lactonase to produce
the open chain monosaccharide 6-phosphogluconate.
3. 6-phosphogluconate is then oxidized and decarboxylated by 6phosphogluconate dehydrogenase to generate ribulose-5P, CO2
and the second molecule of NADPH.
1
2
3
1
2
3
4
The non-oxidative
phase of the PPP
In cells that require high levels
of NADPH for biosynthetic
reactions, the ribulose-5P
produced in the oxidative
phase needs to be converted
back into glucose-6P to
maintain flux through the
glucose-6P dehydrogenase
reaction.
6
5
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.
Where did the 6 carbons go?
1
2
3
4
5
Metabolic flux through the Pentose Phosphate
Pathway is tightly-regulated
1. If increased NADPH is required for biosynthetic pathways, or
to provide reducing power for detoxification, then fructose-6P and
glyceraldehyde-3P are used to resynthesize glucose-6P and
thereby maintain flux through the oxidative phase of the pathway.
2. If cells need to replenish nucleotide pools due to high rates of
DNA and RNA synthesis, then the bulk of ribulose-5P is
converted to ribose-5P to stimulate nucleotide biosynthesis.
3. If ATP levels in the cell are low, and NADPH levels are not
limiting, then glucose-6P dehydrogenase is inhibited and the
pentose phosphate pathway is bypassed so that glucose-6P can
be metabolized directly by the glycolytic pathway.
Regulation of the G6PD activity controls flux through the
glycolytic pathway and pentose phosphate pathways
When the rates of NADPHdependent biosynthetic
reactions are high in the
cytosol, then the
[NADP+]/[NADPH] ratio
increases, leading to
allosteric activation of
glucose-6P dehydrogenase
activity by NADP+ which
increases flux through the
pentose phosphate pathway.
Increased levels of NADPH compete with NADP+ for binding to glucose-6P
dehydrogenase, thereby reducing the activity of the enzyme. This results in
decreased flux through the pentose phosphate pathway and the available glucose6P is then metabolized by the glycolytic pathway to increase production of ATP.
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.
Glutathione reductase uses two electrons from NADPH to
maintain glutathione in the reduced state (GSSG → 2 GSH).
Glucose-6P dehydrogenase deficiency in humans
In erythrocytes, electrons
from glutathione are used
to reduce harmful reactive
oxygen species and
hydroxyl free radicals.
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
Result of observations made 30 years earlier – the anti-malarial drug primaquine
induced acute hemolytic anemia (red blood cell lysis) in a small percentage of
people who had been given primaquine prophylatically. Primaquine inhibits growth
of the malarial parasite in red blood cells by creating a hostile environment
(reactive oxygen species). 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 noncarbohydrate precursors, primarily the carbon backbone of amino acids, plants
use gluconeogenesis to convert GAP to glucose.
•
Three steps in glycolysis must be bypassed by gluconeogenic enzymes in order
to overcome large G differences. Two of the steps simply reverse the reaction
(fructose-1,6-bisphosphatase and glucose-6-phosphatase), whereas, another
step requires two bypass enzymes (pyruvate carboxylase, PEP carboxykinase).
•
Flux through gluconeogenesis and glycolysis is reciprocally-regulated to prevent
futile cycling (burning up ATP). Reciprocal regulation at the PFK-1 (glycolysis)
and F-1,6-BPase (gluconeogenesis) is controlled by the allosteric regulator F2,6-bisphosphate, 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 (lactate, amino acids,
glycerol) for export to other
tissues that depend on glucose
for energy, primarily the brain and
erythrocytes.
Plants use the gluconeogenic
pathway to convert GAP, the
product of the Calvin Cycle, 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 is a mitochondrial enzyme that catalyzes a carboxylation
reaction converting pyruvate to oxaloacetate.
Phosphoenolpyruvate carboxykinase (PEPCK) converts oxaloacetate to
phosphoenolpyruvate (PEP) using the energy released by decarboxylation and
GTP hydrolysis. Transcription of the PEPCK gene is regulated by hormones.
Fructose-1,6-bisphosphatase-1 (FBPase-1) catalyzes the dephosphorylation of
fructose-1,6BP to form fructose-6P; this is the bypass reaction for PFK-1 in
glycolysis.
Glucose-6-phosphatase is an enzyme in liver and kidney cells (not present in
muscle cells) that catalyzes the dephosphorylation of glucose-6P to form
glucose which can be exported out of the cell.
4.Application of
gluconeogenesis
in real life
Pathway Questions
Monitoring blood glucose
levels throughout the day is
critical to diabetics who need
insulin injections.
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.
Glycolysis and gluconeogenesis are
opposing pathways that serve the critical
function of degrading or synthesizing
glucose in response to energy
demands in the cell.
These two pathways share seven of the
same enzymes, with additional pathwayspecific enzymes required at the three
key regulatory steps.
Two of the bypass enzymes in
gluconeogenesis, fructose-1,6bisphosphatase-1 (FBPase-1) and
glucose-6-phosphatase, 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.
Pyruvate carboxylase is a
mitochondrial enzyme that requires
the cofactor biotin to function as a
carboxyl group carrier in a two step
enzyme reaction.
Pyruvate carboxylase is activated
by acetyl CoA and has an important
role in supplying OAA to the citrate
cycle when acetyl CoA levels are
high and the energy charge in the
cell is low.
The cellular location of PEPCK
differs depending on the species.
Humans contain two distinct PEPCK
genes that encode mitochondrial
and cytosolic PEPCK enzymes.
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.
Reciprocal regulation refers to the fact that the same regulatory molecule has
opposite effects on two enzymes that control a shared step in two reaction
pathways. For example, when energy charge in the cell is low, AMP levels are
high leading to activation of PFK-1 (increased flux through glycolysis) and
inhibition of FBPase-1 (decreased flux through gluconeogenesis).
This makes sense
because the pyruvate
generated by glycolysis
can then be used in the
energy conversion
pathways to replenish
ATP, while at the same
time, glucose synthesis is
shutdown resulting in a
build-up of pyruvate.
What is the metabolic logic of reciprocal regulation by citrate?
Reciprocal regulation of PFK-1 and FBPase-1
The allosteric regulator F-2,6-BP is
an even more potent regulator of
these two enzymes than either AMP
or citrate. F-2,6-BP not a metabolic
intermediate in either the
glycolytic or gluconeogenic
pathways, instead it is an allosteric
regulator that activates PFK-1 and
inhibits 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 F-2,6-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 regulated by hormone signaling through
glucagon and insulin which control the activity of a dual function enzyme
containing two catalytic activities, 1) a kinase activity called phosphofructokinase2 (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 and the FBPase-2 activity is inhibited,
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 PFK-2 is inhibited and the activity of FBPase-2 is
stimulated.
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 which leads to phosphorylation of the PFK-2/FBPase-2
enzyme, thereby leading to decreased levels of F-2,6-BP and increased activity of
the gluconeogenic enzyme FBPase-1.
Levels of F-2,6-BP in the cell are controlled by a dual
function enzyme called PFK-2/FBPase-2
In contrast, insulin signaling stimulates protein phosphatase-1 activity resulting
in the dephosphorylation of the PFK-2/FBPase-2 enzyme leading to higher levels
of F-2,6-BP and activation of the glycolytic enzyme PFK-1.
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. Although it costs four high energy phosphate bonds to run the Cori cycle
(the difference between 2 ATP produced by anaerobic glycolysis and 4 ATP and 2
GTP consumed by gluconeogenesis), the benefit to the organism is that glycogen
stores in the muscle can be quickly replenished following prolonged exercise.
.
The Cori Cycle is important for peak performance
Studies on athletes have shown that within 30
minutes of completing a vigorous workout, the
majority of lactate produced during anaerobic
glycolysis in the muscle has been converted
to glucose in the liver and used to replenish
muscle glycogen stores. In fact, the reason
you should "warm down" after exercise (same
movement but under aerobic conditions) is 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.