Carbohydrate Metabolism 1: Pentose Phosphate Pathway, Gluconeogenesis, Reciprocal Regulation

1
Carbohydrate Metabolism 1Pentose Phosphate
Pathway, Gluconeogenesis, Reciprocal Regulation
Bioc 460 Spring 2008 - Lecture 33 (Miesfeld)
primaquine
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
Deficiencies in the enzyme glucose-6P
dehydrogenase affects 400 million people
2
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.

3

• We will cover three primary pathways related to
  carbohydrate metabolism in non-photosynthetic
  organisms
• Pentose phosphate pathway
• Gluconeogenesis
• 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-3-phosphate (GAP) in plants.

4
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

5
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 6-phosphogluconolacto
  ne. 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.

6
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
7
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.

8
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 6-phosphogluconate
  dehydrogenase to generate ribulose-5P, CO2 and
  the second molecule of NADPH.

1
2
3
9
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. 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?
10
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.

11
(No Transcript)
12
Regulation of the G6PD activity controls flux
through the glycolytic pathway and pentose
phosphate pathways
When the rates of NADPH-dependent 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 glucose-6P is then
metabolized by the glycolytic pathway to increase
production of ATP.
13
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).

14
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 GSHGSSG ratio at about 5001.

15
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?
16
Key Concepts Gluconeogenesis

• The importance of gluconeogenesis is to provide
  glucose for cells from non-carbohydrate
  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 F-2,6-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.

17
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.

18
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.Phosphoenolp
  yruvate 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-bisphosphatas
  e-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.

19
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.
20
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 pathway-specific enzymes required at
the three key regulatory steps. Two of the
bypass enzymes in gluconeogenesis,
fructose-1,6-bisphosphatase-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.
21

• 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.

22
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?
23
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.
24
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 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.
25
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.
26
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.
27
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.
28
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.
.
29
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.