Pyruvate Dehydrogenase And TCA cycle Flashcards
What happens to pyruvate before the citric acid cycle?
Before entering the citric acid cycle, Pyruvate derived from glucose and other sugars is oxidized to Acetyl-CoA and Carbon Dioxide by Pyruvate dehydrogenase (PDH)
What is the citric acid cycle?
Citric acid cycle is the final pathway in the oxidation of fuel molecules. Reduced coenzymes go to respiratory chain releasing their electrons and make energy to make ATP
Describe the transport of Pyruvate into the mitochondria
Pyruvate, the end product of aerobic glycolysis freely diffuses through the outer mitochondrial membrane
Pyruvate transporter a carrier protein in the inner mitochondrial membrane transports Pyruvate into the mitochondrial matrix in symporters with protons (H+)
Describe the production of Acetyl-CoA
- Pyruvate is oxidized to Acetyl-CoA and carbon dioxide in the mitochondrial matrix
- Catalyzed by Pyruvate dehydrogenase complex
- irreversible reaction is an oxidative carboxylation
- links citric acid cycle to glycolysis
- NADH formed goes to respiratory chain
Explain the Pyruvate dehydrogenase complex
- Multi enzyme complex containing multiple copies of three enzymes E1, E2 and E3
- Substrate channeling as intermediates they remain bound to enzyme complex during reaction sequence
- Use five coenzymes( 4 derived from vitamins)
- regulated by covalent medication and allosteric effectors
- Also contains two regulatory enzymes, PDH kinase and PDH phosphatase
Explain the E1 coenzyme of PDH complex
E1- thiamine pyrophosphate(TPP) - Vitamin B1(thiamine)
Explain the E2 coenzyme of PDH complex
E2- lipoid acid which is not derived from vitamins
E2- Coenzyme A- pantothenic acid(vitamin B5)
Explain the E3 coenzyme of PDH complex
E3- Flavin Adenine Dinucleotide(FAD) derived from Riboflavin (vitamin B2)
-Nicotinamide adenine dinucleotide (NAD) derived from niacin (vitamin B3)
What is the function of Coenzyme A?
Acts as an acyl carrier
- Has a reactive sulphydryl group that reacts with acyl groups forming thioesters
- Thioesters have a high acyl group transfer potential and so can donate their activated acyl groups to a variety of acceptor molecules
- derived from vitamin B5(pantothenic acid)
Explain the roles of the E1 coenzyme of Pyruvate dehydrogenase complex
E1- Pyruvate dehydrogenase/ decarboxylase- decarboxylates Pyruvate to yield an acyl derivative bound to TPP
Explain the role of E2 coenzymes of Pyruvate dehydrogenase complex
E2- Dihydrolipoyl transacetylase
I. Acyl derivative is oxidized as it is transferred to the oxidized (disulphide) form of lipoid acid
II. The acyl group bound to lipoid acid is transferred to coenzyme A forming Acetyl CoA
Explain the role of E3 coenzymes of Pyruvate dehydrogenase complex
-Dihydrolipoyl dehydrogenase
I. The reduced (sulphylhydryl) form of lipoid acid is oxidized regenerating the disulphide form. FADH2 is formed
II. FADH2 is oxidized to FAD as NAD is reduced to a NADH
Explain the pathway of Pyruvate dehydrogenase complex
- Pyruvate is decarboxylated to form a hydroxyethyl derivative bound to reactive carbon of thiamine pyrophosphate, the coenzyme of Pyruvate decarboxylase (E1)
- The hydroxyethyl intermediate is oxidized by transfer to the disulfide form of lipoic acid covalentlybound to Dihydrolipoyl transacetylase
- The Acetyl group, bound as a thioester to the side chain of lipoic acid, is transferred to CoA
- The sulfhydryl form of lipoic acid is oxidized by FAD-dependent Dihydrolipoyl dehydrogehase(E3), regenerating the disulfide (oxidized) form of lipoic acid
- FADH2 on E3 is reoxidized to FAD as NAD+ is reduced to NADH + H+
What is another name for the citric acid cycle?
Kreb’s cycle or tricarboxylic acd
What is the central pathway energy metabolism where Acetyl groups are oxidized to carbon dioxide
The citric acid cycle
What are the sources of Acetyl CoA?
- The fatty acid, palmitate
- the ketone body, acetoacetate
- the sugar, glucose
- Pyruvate (glucose as well as alanine can be converted to Pyruvate)
- ethanol
What happens as a result of removal of TCA cycle intermediates?
- Removal of TCA cycle intermediates removes the four carbons that are used to regenerate oxaloacetate.
- With depletion of oxaloacetate, it is impossible to continue oxidizing Acetyl-CoA
- Compounds that enter as TCA cycle intermediates replenish the cycle but cannot be fully oxidized to Carbon dioxide
Give an overview of the citric acid cycle
- Two-carbon Acetyl group of Acetyl-CoA is oxidized to two CO2 molecules. P
- Energy released by oxidation is conserved in the reduction of NAD+ and FAD
- The eight electrons donated by the Acetyl group eventually end up in three molecules of NADH and FADH2
- Energy is also conserved as GTP by a substrate level phosphorylation
- Cycle ends with regeneration of 4-carbon acceptor molecule(OAA)
What are the citric acid substrates?
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Substrates:
- Oxaloacetate
- Citrate
- Isocitrate
- a-Ketoglutarate
- Succinyl CoA( SCoA)
- Succinate
- Fumarate
- Malate
What is the overall yield of TCA?
1 TCA cycle revolution produces:
- 3 NADH (9 ATP)
- 1 FADH2 (2 ATP)
- 1 GTP (1 ATP)
12 ATP per Acetyl CoA oxidized
What is the first step to the citric acid cycle?
1- Citrate synthesis
Citrate synthesis catalyzes the irreversible reaction condensation of 2-C Acetyl CoA and 4 Carbon OAA to form citrate (a tricarboxylic acid)
- Gibbs free energy is highly negative, which favors citrate formation - this is one of the regulated steps of TCA cycle
Explain the second step of TCA
2- isomerization of citrate
Aconitase catalyzes the reversible isomerization of citrate to isocitrate
Isocitrate contains a secondary hydroxyl group that can be more readily oxidized
Explain the 3rd step of TCA cycle
3- Oxidation decarboxylation of isocitrate
- Isocitrate dehydrogenase catalyzes the irreversible oxidative decarboxylation of isocitration to a-keto glutarate
- First NADH produced(energy capture)
- The first release of CO2
- One of the rate-limiting steps of the TCA cycle
Explain the 4th step of TCA cycle
oxidative decarboxylation of a-ketoglutarate
- a-ketoglutarate dehydrogenase complex catalyzes the irreversible oxidative decarboxylation of a-ketoglutarate to succinyl-CoA
1. Second NADH produced (energy capture)
2. The second release of CO2
3. One of the regulated steps of the TCA cycle
The large negative Gibbs free energy favors formation of succinyl-CoA
Succinyl CoA is a high energy thioester Like Acetyl CoA
Explain the 5th step of TCA cycle
- Succinate thiokinase (also called succinyl CoA synthetase, named for the reverse reaction)
- A substrate level phosphorylation
- Cleavage of high-energy thioester bond of succinyl CoA is coupled to phosphorylation of GDP to GTP
- GTP and ATP are energetically interconvertible by the nucleoside diphosphate kinase reaction:
GTP + ADP —> GDP + ATP
OR
ATP + GDP —> GTP + ADP(reversible)
What is the 6th step of TCA cycle
- Succinate Oxidation
- Succinate dehydrogenase only enzyme of TCA found in the inner mitochondrial membrane
- Catalyzes the reversible oxidation of Succinate to fumarate
- Electrons are transferred to prosthetic group FAD forming FADH2
-fumarase catalyzes reversible hydration of fumarate to malate
What is the 7th step of TCA cycle
Oxidation of malate
Malate dehydrogenase catalyzes the reversible oxidation of malate to oxaloacetate
1. The third and final NADH of the cycle
2. Gibb’s free energy of reaction is positive, but reaction is driven toward OAA formation by highly exergonic citrate synthase reaction that keeps [OAA] low
Explain the regulation of PDH complex
- In the presence of high energy signals: high ATP, NADH and Acetyl-CoA the PDH complex is turned off(slows down)
- When demand for ATP increases PDH complex is turned on (speeds up)
- In the liver insulin activates the PDH complex, providing Acetyl CoA for fatty acid and cholesterol synthesis
- In the liver glucagon inactivated the PDH complex, saving Pyruvate for gluconeogenesis
Explain the activation of PDH
Dephosphorylation by PDH phosphatase
- Ca 2+ in skeletal muscle - insulin in liver and adipocytes - Catecholamines in cardiac muscle
Increase in energy demand
Increase flux of Acetyl CoA into citric acid cycle
Explain the inhibition of PDH
-product inhibition(allosteric)
- Phosphorylation by PDH-kinase
- glucagon in liver
- Catecholamines in adipocytes
- High energy products (ATP, NADH, and Acetyl-CoA)
- Increased ATP or when sufficient energy is available
Give a general overview of the metabolic effect and clinical features of Pyruvate dehydrogenase deficiency
Metabolic effect:
- increase in Pyruvate with concomitant
- increase in lactic acidosis and alanine(via transamination)
- decrease in production of Acetyl CoA.
- Severe reduction in ATP production
Clinical features: lactic alcohols, neurological defects, myopathy; usually fatal at early age
Explain the vitamin B1 deficiency in the PDH complex
Thiamine deficiency (B1)
Results in Wernicke-Korsakoff syndrome characterized by Ataxia, Opthalmoplegia, memory loss, cerebral hemorrhage
At risk: alcoholics, malnourished individuals
- Heart failure, decrease ATP, increased cardiac output (wet beriberi)
- Other thiamine- requiring enzymes!
a-ketoglutarate DH and branched chain a-ketoacid DH
Who are at risk of thiamine deficiency?
Alcoholics, malnourished individuals
What diseases are the result of thiamine deficiency?
Wernicke-Korsakoff syndrome= ataxia, ophthalmoplegia, memory loss, cerebral hemorrhage
Wet beriberi: increased cardiac output, decrease ATP and heart failure
What is the purpose of the citric acid cycle?
- It ensures that NADH is generated fast enough to maintain ATP homeostasis
- It regulates the concentration of TCA cycle intermediates and their flux into other pathways
What MECHANISMS does regulation of Citric Acid cycle use?
- substrate availability
- product inhibition
- Feedback inhibition primarily by energy status of the cell (ATP/ADP, NADH/NAD)
- Ca2+
List the steps of regulation 9f the citric acid cycle
- Citrate synthesis.
- Isocitrate synthesis
- Alpha-keto glutamate dehydrogenase
- Succinate dehydrogenase
How does citrate synthase regulate TCA cycle?
- substrate availability (Acetyl CoA and oxaloacetate)
- product inhibition by increasing citrate
- inhibited by increased ATP/ADP ratio, increased NADH/NAD+ ratio, increased succinyl CoA and fatty acyl CoA
- activated by increased ADP (or low ATP/ADP )
Explain the regulation of isocitrate
- Inhibited by increased ATP/ADP, increased NADH/NAD+
- inhibition leads to citrate accumulation and transport of excess citrate into cytosol
- Activated by Ca2+ and increased ADP
Explain the regulation of Alpha-ketoglutarate dehydrogenase
- Inhibitted by increased ATP/ADP, increased NADH/NAD+, increased GTP
- product inhibition by increased succinyl-CoA
- Activated by Ca2+
Explain the regulation of Succinate dehydrogenase
- inhibited by OAA
- Activated by ADP, Pi and Succinate
What are the fates of citrate?
- Tricarboxylic acid cycle for energy generation
- Citrate May act as a source of cytosolic reducing equivalents for reductive biosynthesis.
- Carbon source for cytosolic biosynthetic processes, e.g., fatty acids, sterols
- Regulator of other metabolic steps e.g. an inhibitor for phosphofructokinase or Acetyl CoA carboxylase activator
What is the yield of glucose from Acetyl CoA. Formation?
0 to 2 ATP, depending on shuttle that transports electrons from NADH in cytosol
What is the ATP yield from the Kreb’s cycle?
+2 ATP by substrate level phosphorylation
What is the ATP yield from electron transport chain?
About 34 ATP by oxidative phosphorylation
