BioChem Pentose Phosphate Shunt (18)
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Pentose Phosphate Shunt
- otherwise known as the hexose monophosphate or phosphogluconate pathway
- it occurs in the CYTOPLASM; starts with one glycolytic intermediate and ends with another
What are the two major functions of the pentose phosphate shunt?
- 1) biosynthesis of pentose sugars
- (required for synthesis of nucleotides/nucleic acids)
- 2) Formation of NADPH
- (reduced coenzyme required for synthesis of fatty acids, steroids/cholesterol, NTs, nucleotides, regeneration of reduced glutathione, and generation of reactive oxygen)
What are the 3 stages of the pentose phosphate shunt?
- 1) irreversible oxidative conversion of glucose-6-phosphate to pentose-5-phosphate (1 carbon is released as CO2)
- 2) reversible interconversion of 3 pentose-phosphates: ribulose-5-phosphate, xylulose-5-phosphate, ribose-5-phosphate
- 3) reversible conversion of pentose-5-phosphates to glycolytic intermediates!
- glucose-6-phosphate --> 6-phosphoglucono-delta-lactone
- NADP+ is simultaneously reduced to NADPH
What is the major regulatory step in the pentose phosphate pathway?
conversion of glucose-6-phosphate and NADP+ to 6-phosphoglucono-delta-lactone and NADPH by the enzyme glucose-6-phosphate dehydrogenase
- 6-phosphoglucono-delta-lactone --> 6-phosphogluconate
- the lactone is spontaneously hydrolyzed by water, but lactonase increases the rate of hydrolysis
Which pentose phosphate pathway enzyme irreversibly hydrolyzes 6-phosphoglucono-delta-lactone to 6-phosphogluconate?
It is the rapid removal of the _______ product that makes the overall conversion of glucose-6-phosphate to 6-phosphogluconate irreversible
- LACTONE (cyclic ester)
What is the main allosteric inhibitor of glucose-6-phosphate dehydrogenase?
- the in vivo enzyme activity is a small fraction of the potentially maximal velocity b/c the inhibiting NADPH concentration is much higher than that of NADP+ (the substrate)
Which pentose phosphate pathway enzyme catalyzes the oxidative decarboxylation of 6-phosphogluconate to form ribulose-5-phosphate?
What is the main allosteric inhibitor of 6-phosphogluconate dehydrogenase?
Which enzymes generate NADPH in the pentose phosphate pathway?
- glucose-6-phosphate dehydrogenase
- 6-phosphogluconate dehydrogenase
- oxidatively decarboxylates 6-phosphogluconate to form ribulose-5-phosphate (it's a ketose)
- the enzyme uses NADP+ as a coenzyme, therefore NADPH is formed in addition to the ribulose
- also CO2 is released
- the reaction is IRreversible, and inhibited by the product NADPH
What are the possible fates of ribulose-5-phosphate once it's been synthesized from the oxidative phase of the pentose-phosphate shunt?
- it can be converted into other pentose-phosphates, eg:
- 1) xylulose-5-phosphate: by phosphopentose epimerase (ribulose & xylulose = epimers)
- 2) ribose-5-phosphate: by phosphopentose isomerase
- converts ribulose-5-phosphate to xylulose-5-phosphate
- ribulose and xylulose are epimers
converts ribulose-5-phosphate to ribose-5-phosphate
True or False: in the pentose phosphate pathway, the conversion of ribose-5-phosphate to glycolytic intermediates generates NADPH?
- FALSE, only the first stage of the pentose phosphate pathway (from glucose-6-phosphate to ribulose-5-phosphate) generates NADPH
- the reversible part of the shunt allows for both the degradation of pentose-phosphates & the synthesis of pentose-phosphate from glycolytic intermediates WITHOUT production of NADPH
- enzyme that uses coenzyme TPP to transfer a 2-carbon fragment FROM a ketose (xylulose-5-phosphate) TO an aldose (ribose-5-phosphate)
- products = 7-carbon ketose & 3-carbon glyceraldehyde-3-phosphate
After reacting with transketolase, xylulose is now ___________ and ribose is now ___________.
- xylulose --> glyceraldehyde-3-phosphate
- ribose --> sedoheptulose (7-carbon ketose)
- takes two carbons from xylulose and puts them on ribose
What coenzyme is required for transketolase activity?
thiamine pyrophosphate (TPP)
Measuring the activity of which pentose phosphate pathway enzyme can be used to assay for thiamine deficiency?
How many carbons are in the fragments transketolase transfers from a ketose to an aldose?
2 carbons (think transkeTWOlase)
- transfers 3-carbon fragment from sedoheptulose(a 7-carbon ketose) to glyceraldehyde-3-phosphate (an acceptor 3-carbon aldose)
- products: a residual erythrose-4-phosphate (4-C)
- & fructose-6-phosphate (6-C)
- aldol condensation or reverse aldol condensation
After reacting with transaldolase, sedoheptulose is now _________ and glyceraldehyde-3-phosphate becomes _________.
- sedoheptulose --> erythrose-4-phosphate
- glyceraldehyde-3-phosphate --> fructose-6-phosphate
- takes 3 carbons from sedoheptulose and puts them on glyceraldehyde-3-phosphate
How many carbons are in the fragments transaldolase transfers from a ketose to an aldose?
What is the other carbon skeleton rearrangement reaction transketolase catalyzes?
- transfer of a 2-carbon fragment from xylulose-5-phosphate (5-C) to erythrose-4-phosphate (4C)
- products = fructose-6- phosphate (6 carbons) and glyceraldehyde-3-phosphate (3 carbons)
- TPP again serves as coenzyme
What is the overall result of the rearrangements catalyzed by transketolase and transaldolase?
- one glyceraldehyde-3-phosphate
- two fructose-6-phosphates
- from three pentose-phosphates, which were formed from 3 molecules of glucose-6-phosphate
What & how many glycolytic intermediates are produced by the rearrangement of three ribulose-5-phosphate molecules by transketolase and transaldolase?
two fructose-6-phosphate molecules and one glyceraldehyde-3-phosphate molecule are produced
What is the net balanced equation of the pentose phosphate pathway?
3 glucose-6-phosphate + 6 NADP --> 2 fructose-6-phosphate + glyceraldehyde-3-phosphate + 3 CO2 + 6 NADPH + 6 H+
What is the structural difference between NADH and NADPH?
- NADPH has an additional phosphate group at the 2'-position of the ribose linked to adenine that allows enzymes to discriminate between NADH and NADPH (specificity handle)
- NADP+ still accepts a pair of electrons & a proton in the nicotinamide ring like NAD+
If the NAD+/NADH ratio in a cell is very high, the NADP+/NADPH ratio in the same cell is:
Very LOW: a cell with a high concentration of NAD+ will therefore also have a high concentration of NADPH
NADPH __ NADP+
NAD+ __ NADH
- NADPH is in much higher concentrations than NADP+ (NADPH >> NADP+)
- On the other hand, NAD+ is in much higher concentrations than NADH (NAD+ >> NADH)
- NADPH provides reducing power for biosynthetic work OUTSIDE the mitochondria
What are the tissues in which the pentose phosphate pathway is most active?
- places where NADPH is used:
- Liver (coenzyme for drug-metabolizing enzymes)
- Adipose tissue (for synthesis of fatty acids/steroids)
- adrenal cortex (for steroid hormone synthesis)
- erythrocytes (protects from oxidative damage)
- phagocytes (as substrate for NADPH oxidase that protects against neutrophil infection via respiratory bursts)
How can the pentose phosphate pathway be manipulated if you're under extreme stress (eg. oxidative attack)?
- you'd want to make a lot of NADPH, so the shunt would be run to make NADPH, then the resulting product would be re-converted back into glucose-6-phosphate and the shunt would be run run again until all carbons you started with were exhausted
- this is the most extreme form of needing the pathway
How can the pentose phosphate shunt be regulated?
- glucose-6-phosphate dehydrogenase is the first committed step AND the rate limiting step (will only proceed if energetically favorable)
- insulin induces the pathway
- NADPH inhibits the pathway (allosteric feedback)
- generates a superoxide anion from oxygen
- the generation of reactive oxygen species (ROS) by phagocytes is an important mechanism used to kill engulfed bacteria
What are examples of reactive oxygen species'?
superoxide, hydrogen peroxide & hydroxyl radicals
enzyme that generates hypochlorite (bleach) from hydrogen peroxide and chloride anions during the oxidative burst of a neutrophil
congenital granulomatous disease
- defects in forming ROS' (most importantly, the superoxide radical)
- can result in the patient being subject to severe recurrent infection + chronic inflammatory conditions due to an inability to kill ingested pathogens
What are some ways reactive oxygen can be harmful? (4)
- can cause damage to DNA, especially mtDNA
- lipid peroxidation causes increases in permeability that change ion concentration across membranes
- it can attack some amino acids and promote cross-linking or fragmentation
- they can form in damaged cells/tissues, eg. those recovering from ischemia (problem associated with myocardial infarction, bypass surgery, transplantation)
superoxide dismutase (SOD)
- detoxifies reactive oxygen species'
- takes 2 superoxide anions + 2 protons --> hydrogen peroxide (H2O2) + oxygen (O2)
enzyme that catalyzes the decomposition of hydrogen peroxide into water and oxygen
family of enzymes that reduce peroxides by catalyzing the transfer of electrons from an electron donor to the peroxide
glutathione peroxidase (GSH peroxidase)
- GSH peroxidase reduces hydrogen peroxide to two water molecules
The tripeptide glutathione is an important coenzyme for which enzyme?
- glutathione peroxidase (GSH peroxidase), an enzyme that converts hydrogen peroxide to water
- as the peroxide is reduced, glutathione is oxidized
The NADPH produced in the pentose phosphate pathway plays what role in the reduction of hydrogen peroxide to water by GSH peroxidase?
- As GSH peroxidase reduces peroxide, it oxidizes the coenzyme glutathione
- NADPH produced by glucose-6-phosphate dehydrogenase is used to regenerate the reduced form of glutathione (to further eliminate other peroxide)
- hydroxyl radicals are generated from hydrogen peroxide via Fe (iron) oxidation
- generates •OH (hydroxyl radical) from H2O2 (hydrogen peroxide) and superoxide (•O2-)
- is a possible source for oxidative stress
What is the main function of the pentose phosphate pathway in erythrocytes?
to make NADPH which plays a role in protecting erythrocytes from oxidative damage
defective glucose-6-phosphate dehydrogenase (G6PD)
- enzyme that catalyzes NADPH formation
- when individuals w/ a defect are exposed to oxidants (from food or drugs) they cause denaturation/precipitation of hemoglobin within RBCs
- this makes RBCs inflexible and subject to damage as they pass through narrow capillaries
- breakdown of RBCs in the spleen leads to anemia & release of hemoglobin (which may be excreted in urine)
- evidence of how NADPH plays an important role in detoxifying reactive oxygen & especially protecting red blood cells from oxidative damage
Which chromosome is the gene for glucose-6-phosphate dehydrogenase (G6PD) located on?
the X chromosome
When someone with a glucose-6-phosphate dehydrogenase (G6PD) deficiency takes primaquine what condition develops?
severe acute hemolytic anemia
A patient is developing severe acute hemolytic anemia after taking the antimalarial drug primaquine. What enzyme is likely deficient?
glucose-6-phosphate dehydrogenase (G6PD)
Why do gene mutations that affect the Km or Ki of glucose-6-phosphate dehydrogenase (G6PD) make a person more susceptible to acute hemolytic anemia than changes in the enzyme's Vmax?
- because the Km and Ki determine whether the in vivo activity of the enzyme can be increased to meet an oxidative challenge
- the in vivo activity is only a small fraction of the potential Vmax
- if the Km and Ki of a mutant enzyme are such that the enzyme does not respond to a change in the NADP+/NADPH ratio in the in vivo range, severe oxidative damage and red cell destruction ensues
What are some foods/classes of drugs that can trigger episodes of hemolytic anemia in people with glucose-6-phosphate dehydrogenase deficiency?
Fava beans, antimalarial drugs, some antibiotics & antipyretics
What are some disease states that are caused by free radical injury? (6)
- Acute Renal Failure
- Neurodegenerative disease
- an enzyme NOT found in erythrocytes that produces NADPH in a pathway other than the pentose phosphate shunt
- Malate + NADP+ --> Pyruvate + CO2 + NADPH + H+
- also known as NADP-linked malate dehydrogenase
Why are erythrocytes at a greater risk of oxidative damage than other cells?
because RBCs lack alternative ways to generate NADPH, such as malic enzyme
What are the substrates and products of the reaction catalyzed by malic enzyme?
Malate + NADP+ --> Pyruvate + CO2 + NADPH + H+
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