Download METABOLISM IN HEALTH AND DISEASES I Lecture 2 Pentose

BCHEM 254
METABOLISM IN HEALTH
AND DISEASES II
Lecture 2:
Pentose Phosphate Pathway
Lecturer:
Christopher Larbie, PhD
• In most animal tissues, glucose is catabolized via the
glycolytic pathway into two molecules of pyruvate.
• Pyruvate is then oxidized via the citric acid cycle to
generate ATP.
• There is another metabolic fate for glucose used to
generate NADPH and specialized products needed by the
cell.
• This pathway is called the pentose phosphate pathway.
• Some text books call it the hexose monophosphate
shunt, still others call it the phosphogluconate pathway.
• The pentose phosphate pathway produces NADPH which
is the universal reductant in anabolic pathways
• In mammals the tissues requiring large amounts of NADPH
produced by this pathway are the tissues that synthesize
fatty acids and steroids such as the mammary glands,
adipose tissue, adrenal cortex and the liver.
• Tissues less active in fatty acid synthesis such as skeletal
muscle are virtually lacking the pentose phosphate
pathway.
• The second function of the pentose phosphate pathway is
to generate pentoses, particularly ribose which is necessary
for the synthesis of nucleic acids.
The Pentose Phosphate Pathway (PPP
• It is convenient to think of the pentose phosphate pathway
as operating in two phases.
• The first phase is the oxidative phase. Two of the first
three reactions of the first phase generate NADPH. The
second phase is the nonoxidative phase.
• In the first step glucose-6-phosphate is oxidized into
ribulose-5-phosphate and CO2.
• During the oxidation of glucose-6-phosphate, NADP+ is
reduced into NADPH.
• The second step of the pathway coverts the ribulose 5-
phosphate into other pentose-5- phosphates including
ribose-5-phosphate used to synthesize nucleic acids.
• The third step includes a series of reactions that convert
three of the pentose-5-phosphates into two molecules of
hexoses and one triose.
• In the fourth step, some of these sugars are converted into
glucose-6-phosphate so the cycle can be repeated.
• The direction of the pathway varies to meet different
metabolic conditions.
Oxidative Phase
• The oxidative phase of the pentose phosphate pathway is
composed of three steps;
1. Glucose-6-Phosphate dehydrogenase (G6PD)
•
•
reaction:
The PPP begins with the oxidation of glucose-6phosphate to produce a cyclic ester (the lactone of
phosphogluconic acid) and NADPH.
G6PD is highly specific for NADP+. This reaction is
irreversible and highly regulated by NADPH and fatty
acid esters of coenzyme A (which are intermediates of
fatty acid biosynthesis).
• Inhibition by NADPH is dependent on the cytosolic
NADP+/NADPH ratio (in the liver it’s about 0.015).
2. Gluconolactonase reaction:
• The gluconolactone produced in step 1 is hydrolytically
unstable and readily undergoes a spontaneous ringopening hydrolysis, although the enzyme accelerates the
reaction
• The product is 6-phospho-D-gluconate, a sugar acid which
is further oxidized in the next step
3. 6-phosphogluconate dehydrogenase reaction
• This enzyme catalyses the oxidative decarboxylation of 6phosphogluconate to yield D-ribolus-5-phosphate and
NADPH.
• This reaction occurs in two distinct steps:
• The initial NADP+-dependent dehydrogenation yields a β-
keto acid, 3-ketp-6-phosphogluconate, which is very
susceptible to decarboxylation (the second step).
• The product of the reaction, riboluso-5-phosphate is the
substrate for the non-oxidative reactions.
Nonoxidative Phase
• The nonoxidative phase of the pentose phosphate
pathway is composed of 5 steps but only 4 types of
reactions.
• This portion of the pathway begins with an isomerization
and epimerization, and it leads to the formation of either Dribose-5-phosphate or D-xylulose5-phosphate.
• These intermediates can then be converted into glycolytic
intermediates or directed to biosynthetic processes
4. Phosphopentose Isomerase Reaction
• This enzyme converts ribulose-5-phosphate and ribose-5phosphate via an edediol intermediate.
• The ribose-5-phosphate produced in this reaction is utilized
in the biosynthesis of coenzyems (including NADH,
NADPH, FAD and B12), nucleatides and nucleic acids
(DNA and RNA). The net reaction to this point is
Glucose-6-phosphate + 2NADP+ → ribose-5-phosphate +
CO2 + 2NADPH + 2H+
5. Phosphopentose Epimerase Reaction
• This enzyme converts riboluse-5-phosphate to another
ketose, xylulose-5-P proceeded by an enediol
intermediate at C-3.
• In the reaction, an acidic proton located α- to a carbonyl
carbon is removed to generate the enediolate, but the
proton is added back to the same carbon from the
opposite side.
• From the beginning to this point, 2 NADPH has been
generated or every molecule of glucose-6-P converted to
pentose-5-P
• The next three steps, there will be rearrangement to
generate three-, four-, six- and seven-carbon units, which
can be utilized in various metabolic processes
• This is important because, very often the cellular need for
NADPH is considerably greater than the need for ribose5-P
• The next three steps thus return some of the 5-C units to
glyceraldehyde-3-P and fructose-6-P, which can enter the
glycolytic pathway
• The advantage of this is that the cells have met its need
for NADPH and ribose-5-phosphate in a single pathway,
yet at the same time it can return excess carbon
metabolites to glycolysis
6. Transketolase Reaction
• This enzyme catalysis the transfer two carbon unit from
xylulose-5-P to ribose-5-P yielding 3-C unit glyceraldehyde-3P and sedoheptulose-7-P
• The donor molecule is a ketone and the acceptor molecule is
an aldose. G-3-P enters the glycolytic pathway.
• Transketolase is dependent on thiamine pyrophosphate as a
cofactor.
• The mechanism involves the abstraction of the acidic thiozole
proton of TPP, attack by the resulting carbanion at the
carbonyl carbon of the ketose phosphate substrate, expulsion
of the G-3-P product, and transfer of the 2 C unit.
• Transketolase can process a variety of 2-keto sugar
substrates in a similar manner as in reaction 8
7. Transaldolase Reaction
• This enzyme catalysis the conversion of S-7-P and G-3-P to
erthrose-4-P and fructose-6-P.
• This reaction is similar to the aldose reaction of glycolysis. It
involves the formation of a Schiff base intermediate between the
S-7-P and an active-site lysine residue.
• Elimination of the E-4-P product leaves an enamine of
dihydroxyacetone, which remains stable at the active site until the
other substrate comes into position.
• Attack of the enamine carbanion at the carbonyl of G-3-P is
followed by hydrolysis of the Schiff base (imine) to yield the
product fructose-6-phosphate.
8. Transketolase Reaction
• This reaction is similar to Step 6. Here the transketolase
catalysis the transfer of a 2C unit from X-5-P to E-4-P
yielding G-3-P and fructose-6-P.
• Both products enter the glycolytic pathway
Net Reaction
• Oxidative phase:
• 3 Glucose-6-phosphate + 6 NADP+ → 2 Xylulose-5p +
ribose-5-P + 3CO2 + 6NADPH + 6H+
• Rearrangements of the non-oxidative phase:
• 2 Xylulose-5-P + ribose-5-P → 2 Frucose-6P +
Glyceraldehyde-3-P
• The sum of these two phases:
• 3 Glucose-6-phosphate + 6 NADP+ →2 Frucose-6-P +
Glyceraldehyde-3-P + 3 CO2 + 6 NADPH + 6 H
Tailoring the Pentose Phosphate Pathway to meet
specific needs of the cell
1. If the cell requires both ribose-5-P and NADPH, the
first four reactions of the PPP predominate.
• NADPH is produced by the oxidative reactions of the
pathway, and ribose-5-P is the principal product of carbon
metabolism
2. More ribose-5 phosphate needed than NADPH
• This can be can be accomplished with the synthesis of
ribose-5-P with NADPH by by-passing the oxidative steps.
• This is achieved by the withdrawal of F-6P and G-3-P, but
not glucose-6-P, from glycolysis.
• The reaction of transketolase and transaldolase on F-6-P
and G-3-P produces three molecules of ribose-5-P from two
molecules of F-6-P and one G-3-P.
• In this route, no carbon metabolites return to glycolysis.
5 G-6-P + ATP → 6 R-5-P + ADP + Pi
3. More NADPH needed than Ribose-5-phosphate
• Large amounts of NADPH can be supplied in the PPP without
concomitant production of ribose-5-P if ribose-5-P produced in the
PPP is recycled to produce glycolytic intermediates.
• This alternative involves a complex interplay between the
transketolase and transaldolase reactions to convert ribulose-5-P to
F-6-P by gluconeogenesis.
• The net reaction is
6 G-6-P + 12 NADP+ + 6 H2O → 6 Ribulose-5-P + 6 CO2 + 12 NADPH
+ 12 H+
6 Ribulose-5-P → 5 Glucose-6-P + Pi
Net: Glucose-6-P + 12 NADP+ + 6 H2O → 6 CO2 + 12 NADPH + 12H+
+ Pi
4. Both NADPH and ATP are needed but not
ribose-5-phosphate
• Under some conditions, both NADPH and ATP must be
provided in the cell.
• This can be accomplished in a series of reactions similar in
case 3 if the F-6-P and G-3-P produced in this way proceed
through glycolysis to produce ATP and pyruvate, which itself
can yiled even more ATP by continuing on to the TCA cycle.
• The net reaction for this alternative is
3 Glucose-6-P + 5 NAD+ + 6 NADP+ + 8 ADP + 5 Pi → 5
Pyruvate + 3 CO2 + 5 NADH + 6 NADPH + 8 ATP + 2 H2O + 8
H+
Regulation
• The first step of the phosphopentose pathway is the
irreversible committed step.
• This reaction is catalysed by glucose-6-phosphate
dehydrogenase
• This step is of course allosterically regulated. The product
of this reaction NADPH is a strong inhibitor
• So when the cytosol concentration of NADPH is high, the
enzyme’s activity is low. It is also allosterically regulated by
fatty acid acyl esters of coenzyme A
• The transcription of the gene for this enzyme is under
hormonal regulation
• Xylulose-5-P also serves as a signalling molecule.
• When blood glucose levels rise, glycolysis and PPP pathways
are activated in the liver and the X-5-P produced in the latter
pathways stimulate protein phosphate 2A (PP2A)
• The (PP2A) dephosphorylate the bifunctional enzyme
phosphofructokinase-2 or fructose-2,6-bisphosphatase
• Increased levelf of F-2,6-BP stimulate glycolysis and inhibit
gluconeogenesis.
• At the same time, PP2A dephosphorylates carbohydrate
responsive element –binding protein (ChREBP), a
transcription factor that activates expression of liver genes for
lipid synthesis.
• These effects are a powerful combination. Increased
glycolysis produces substantial amounts of acetyl-CoA,
the principal substrate for lipid synthesis.
• The PPP produced NADPH, the source of electrons for
lipid biosynthesis.
• Elevated expression of the appropriate genes sets the
stage for lipid biosynthesis in the liver, an important
consequence of ingestion of carbohydrates
Glucose-6-phosphate dehydrogenase deficiency
• Glucose-6-phosphate dehydrogenase deficiency (G6PD) is
an X-linked recessive hereditary disease characterized by
abnormally low levels of glucose-6-phosphate
dehydrogenase, especially important in red blood cell
metabolism.
• G6PD deficiency is the most common human enzyme
defect.
• Individuals with the disease may exhibit non-
immune haemolytic anaemia in response to a number of
causes, most commonly infection or exposure to certain
medications or fava beans.
• Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme in
the pentose phosphate pathway.
• G6PD converts glucose-6-phosphate into 6-phosphogluconoδ-lactone
• This is the rate-limiting enzyme of this metabolic pathway that
supplies reducing energy to cells by maintaining the level of
the co-enzyme nicotinamide adenine dinucleotide
phosphate(NADPH)
• The NADPH in turn maintains the supply of
reduced glutathione in the cells that is used to mop up free
radicals that cause oxidative damage.
• The G6PD / NADPH pathway is the only source of reduced
glutathione in red blood cells (erythrocytes)
• The role of red cells as oxygen carriers puts them at substantial
risk of damage from oxidizing free radicals except for the
protective effect of G6PD/NADPH/glutathione
• People with G6PD deficiency are therefore at risk of haemolytic
anaemia in states of oxidative stress
• Oxidative stress can result from infection and from chemical
exposure to medication and certain foods.
• Broad beans, e.g., fava beans, contain high levels
of vicine, divicine, convicine and isouramil, all of which
are oxidants
• When all remaining reduced glutathione is consumed,
enzymes and other proteins (including haemoglobin) are
subsequently damaged by the oxidants, leading
to electrolyte imbalance, cross-bonding and protein
deposition in the red cell membranes.
• Damaged red cells are phagocytosed and sequestered
(taken out of circulation) in the spleen.
• The haemoglobin is metabolized
to bilirubin (causing jaundice at high concentrations).
• The red cells rarely disintegrate in the circulation, so
haemoglobin is rarely excreted directly by the kidney, but this
can occur in severe cases, causing acute renal failure
• Deficiency of G6PD in the alternative pathway causes the
build-up of glucose and thus there is an increase
of advanced glycation end-products (AGE)
• The deficiency also reduces the amount of NADPH, which
is required for the formation of nitric oxide (NO)
• Although female carriers can have a mild form of G6PD
deficiency (dependent on the degree of inactivation of the
unaffected X chromosome), homozygous females have
been described
• In these females there is co-incidence of a rare immune
disorder termed chronic granulomatous disease (CGD)