Download Lecture 33 Carbohydrates1

Lecture 33
Carbohydrate Metabolism 1
Key Concepts
• Pentose Phosphate Pathway
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Enzymatic reactions in the oxidative phase
Enzymatic reactions in the nonoxidative phase
Glucose-6P dehydrogenase deficiency in humans
NADPH vs. NAD+ in Metabolism
• Gluconeogenesis and the Cori Cycle
– Review of gluconeogenesis
– The Cori Cycle provides glucose to muscle cells during exercise
•
What is the biochemical basis for favism and how is it related to
malarial resistance?
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How does fructose-2,6-bisphosphate control flux through
gluconeogenesis and glycolysis?
Three primary pathways in
anabolic carbohydrate
metabolism in nonphotosynthetic 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
Overview of the Pentose Phosphate Pathway
•
Takes place entirely within the cytoplasm
•
Also known as:
– the hexose monophosphate shunt
– phosphogluconate pathway
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The most important function:
– to reduce two molecules of NADP+ to NADPH for each glucose-6phosphate (glucose-6P) that is oxidatively decarboxylated to
ribulose-5-phosphate (ribulose-5P)
– also responsible for producing ribose-5-phosphate (ribose-5P)
from glucose-6P
Overview of roles of NAD+ vs. NADP+
Two important conjugate redox pairs:
NAD+/NADH vs. NADP+/NADPH
NAD+ functions as the primary oxidant in the cell
(accepts electrons)
NADPH is the primary reductant in the cell
(donates electrons)
[NAD+]/[NADH] ratio is close to 1,000
[NADP+]/[NADPH] ratio is 0.01
Two Phases of Pentose Phosphate Pathway
oxidative phase
–
generates NADPH
nonoxidative phase
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–
interconverts C3,
C4, C5, C6 and C7
sugar phosphates
uses many of the
same "carbon
shuffle" reactions
we saw in the Calvin
cycle.
Flux through Pentose Phosphate Pathway is
Tightly Regulated
Regulated in response to:
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–
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the NADP+/NADPH ratio
energy needs of the cell
requirements for nucleotide and coenzyme biosynthesis
1. When NADPH is needed, ribulose-5P is converted back into glucose6P to maintain flux through the pathway.
2. If ATP and NADPH are needed (which would be the case for most
anabolic pathways), then some of the ribulose-5P is used to synthesis
hexose phosphates for glycolysis.
3. If the cell needs to increase the rate of nucleotide and coenzyme
biosynthesis, then most of the ribulose-5P is shunted toward ribose-5P
synthesis.
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)– 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 glucose6P 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 and by compounds in fava beans.
http://www.g6pd.org/favism/english/index.mv
Dish of Broad Beans by Giovanni Garzoni
Enzymatic reactions in the oxidative phase
Three enzymatic reactions:
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. The commitment
step in the pathway.
2. 6-phosphogluconolactone is hydrolyzed by lactonase to
produce the open chain monosaccharide 6phosphogluconate
3. 6-phosphogluconate is then oxidized and
1
2
3
decarboxylated
by 6-phosphogluconate
dehydrogenase to generate ribulose-5P, CO2 and the
second molecule of NADPH.
Enzymatic reactions in the nonoxidative phase
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 that
we saw in the Calvin cycle.
Let’s Play: Count the Carbons!
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1
2
2
3
3
4
5
6
4
5
Six C5 molecules (ribose-5P and xylulose-5P) are used to resynthesize five C6
molecules (glucose-6P) in the nonoxidative phase.
Glucose-6P is a substrate for both the glycolytic pathway
and the pentose phosphate pathway
What controls the flux???
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
glucose-6P is then metabolized by the glycolytic pathway as a source of
energy for the 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 keep
cysteine residues in
hemoglobin in the reduced
state, and for reducing 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.
An active pentose phosphate
pathway in erythrocytes
normally provides sufficient
levels of NADPH to maintain
the GSH:GSSG ratio at about
500:1.
Discovery of G6PD deficiency – Mid 1950s
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. The
biochemical basis for this druginduced illness was found to be
lower than normal levels of
NADPH in erythrocytes due to a
G6PD deficiency.
The same acute hemolytic anemia
seen in individuals with G6PD
who are treated with primaquine
explains the symptoms of favism.
One of the active compounds in
fava beans is called vicine, a toxic
glycoside that induces oxidative
stress in erythrocytes.
Overview of Gluconeogenesis and the Cori Cycle
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When dietary sources of glucose are insufficient, and glucose stores
have been depleted, glucose is synthesized from non-carbohydrate
compounds by a series of cytosolic reactions called the
gluconeogenic pathway.
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We have seen this pathway before.
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Gluconeogenesis converts pyruvate to glucose using a set of
reactions that require energy input in the form of ATP and GTP.
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Importantly, gluconeogenesis is not simply the reversal of
glycolysis.
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The Cori Cycle provides glucose to muscle cells during exercise
Common to Both
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
Pathway Questions
3. What are the key enzymes in gluconeogenesis?
Pyruvate carboxylase – is a mitochondrial enzyme that catalyzes a
carboxylation reaction converting pyruvate to oxaloacetate using a reaction
mechanism involving a biotinyl "swinging arm" and ATP hydrolysis. Pyruvate
carboxylase is dependent on allosteric activation by acetyl CoA.
Phosphoenolpyruvate carboxykinase (PEPCK) – is localized to either the
mitochondrial matrix or the cytosol (or both in the case of human liver cells) and
catalyzes a phosphoryl transfer reaction that 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. FBPase-1 is inhibited by the allosteric
regulators F2,6BP and AMP which are also allosteric activators of PFK-1.
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. Glucose-6-phosphatase is
located in the lumen of the endoplasmic reticulum.
Review Control of Glycolysis
vs. Gluconeogenesis
You should understand:
Reciprocal control of glycolysis and
gluconeogenesis
Regulation of PFK-1 and FBPase-1
Pyruvate carboxylase and
phosphoenolypyruvate
carboxykinase (PEPCK),
are required to catalyze the
bypass reaction that
converts pyruvate to PEP.
Pyruvate carboxylase is
activated by acetyl CoA and
has an important role in
feeding OAA to the citrate
cycle when acetyl CoA
levels are high and the
energy charge in the cell is
low.
Humans have two distinct PEPCK genes that encode
mitochondrial and cytosolic PEPCK enzymes
Moving NADH
equivalents to
cytosol
Oxidation of lactate by
lactate dehydrogenase
Regulation of PFK-2/FBPase-2
The Cori Cycle
Both pathways (glycolysis
and gluconeogenesis)
are active at the same
time, but in different cells.
Can be quite
advantageous.
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 costs four high energy phosphate bonds, however, the
benefit to the organism is that glycogen stores in the muscle can be quickly
replenished following prolonged exercise.
The reason athletes 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.
For more info (relatively accurate): http://www.cytosport.com/science/lacticacid.html
Just don’t buy the guy’s juice!