5 - Citric Acid cycle Flashcards
What is the purpose of the TCA cycle? What do carbs, proteins and lipids enter as? How is the process amphibolic?
Oxidative pathway by which carbs, lipids and proteins are catabolized in aerobic systems (mitochondrial matrix)
Carbs: Pyruvate
Protein: TCA cycle intermediates of amino acid metabolism
Lipids: acetyl-CoA
Amphibolic: catabolic (first part - oxidation to produce CO2) anabolic (second part - regeneration of oxaloacetate)
List the coenzymes involved in the TCA cycle
Thiamine pyrophosphate (TPP) - Decarboxylations of alpha-ketoacids
Lipolic acid as lipoamide
- Acyl group and electron carrer
Flavin coenzymes (FAD, FMN) - Electron transfer reactions via reduction of isoalloxazine ring system
Coenzyme A (patnothenic acid and beta mercaptoethylamine)
- Free energy of thioester hydrolysis is high relative to oxyester
- Nucleophilic properties of the thioalkoxide anion
- Activation and transfer of acetyl and acyl groups
Describe the pyruvate dehydrogenase complex
- Large and complex (3 enzymes and 5 coenzymes)
3 enzymes:
- Pyruvate dehydrogenase
- Dihydrolipoamide transacetylase
- Dihydrolipoamide dehydrogenase
Give the six steps for the pyruvate dehydrogenase catalyzed conversion of pyruvate to acetyl-CoA
- TPP carbanion of E1 mediates the elimination of CO2 from pyruvate and reduction of carbonyl to alcohol.
- 2C alcohol is transfered from E1 TPP to E2-Lip1 and oxidized to acetate.
- E2-Lip1 acetate is transfered to E2- Lip2.
- Acetate is released from E2-Lip2 as acteyl-CoA.
- E2-Lip2 is oxidized to disulfide and E3 FAD is reduced to FADH2.
- E3 FADH2 is re-oxidized to FADH and NAD+ is reduced to NADH. Transfer of reducing equivalents outside of the PDC complex
Free energy of overall reaction is -33.5 kJ/mole
What are the benefits of molecular compartmentalization?
- Allows substrate channelling
- Increases probability of enzyme-substrate encounter
- Allows for coordinate regulation of multiple enzymes
Summarize the three types of reactions in the TCA cycle
1 GTP generating reaction (succinyl-CoA synthetase)
2 dehydrogenase eliminations of CO2 (isocitrate DH and alpha-ketoglutarate DH)
4 dehydrogenase generations of reduced electron carriers
- 3 NADH (isocitrate DH, alpha-ketoglutarate DH, malate DH)
- 1 FADH2 (succinate dehydrogenase)
List the main metabolites of the TCA cycle in the order that they ‘appear.’
- Acetyl -CoA + oxaloacetate
- citrate
- isocitrate
- Alpha-ketoglutarate
- succinyl-CoA
- succinate
- fumarate
- Malate
- Oxaloacetate
List reactions 1-4 (intro and loss of two carbon atoms) of the TCA cycle
- Citrate synthase reaction- first committed step and site of regulation of the overall pathway; condensation reaction with methyl carbon of acetyl-CoA acting as nucleophile in attack on carbonyl carbon of oxaloacetate; intermediate spontaneously hydrolyses to citrate
- Aconitase reaction- isomerization reaction involves sequential dehydration and then hydration of citrate; consumption of the isocitrate product drives the aconitase reaction to the right
- Isocitrate dehydrogenase reaction- dehydrogenation to oxalosuccinate that spontaneously decarboxylates to -ketoglutarate; dehydrogenation requires NAD+
- -Ketoglutarate
dehydrogenase complex reaction- analogous to the pyruvate dehydrogenase reaction; uses same 5 coenzymes and a multisubunit enzyme complex; first step is decarboxylation using TPP remaining reactions are same as pyruvate dehydrogenase steps but regulatory activities are not present; product of the complex reaction is succinyl-CoA

List reactions 5-8 (generation of oxaloacetate) of the TCA cycle
- Succinyl-CoA synthetase reaction- potential energy in succinyl-CoA is captured in conversion of GDP to GTP; this is then used by nucleoside diphosphokinase to generate ATP from ADP
- Succinate dehydrogenase reaction - FAD-dependent dehydrogenation to form fumarate; FAD is more powerful oxidant than NAD and required for the C-C oxidation; occurs on the enzyme bound to the inner mitochondrial membrane; important for coupling of the FADH2 oxidation via the electron transport system (also on membrane) enzyme is stereoselective for trans-isomer product (i.e. fumarate not maleate)
- Fumarase reaction- hydration of the C-C double bond to form malate stereospecific in both directions
- Malate dehydrogenase reaction - dehydrogenation of alcohol to ketone (oxaloacetate); NAD+ dependent reaction; reaction driven to product by consumption of oxaloacetate in the citrate synthase reaction

List the overall reaction of the TCA cycle
Acetyl-CoA + 3H2O + 3NAD+ + FAD + GDP + Pi -> 2CO2 + 3NADH + FADH2 + CoA-SH + GTP
What is the total ATP yield from glucose
Reaction from glycolysis to pyruvate (x2) through citric acid cycle:
Glucose + 2H2O + 10NAD+ + 2FAD + 4ADP + 4Pi -> 6CO2 +10NADH+6H+ +2FADH2 +4ATP
Most of the remaining energy is captured during the reoxidation of NADH and FADH2 in the respiratory chain (3 moles ATP per mole NADH; 2 moles ATP per mole FADH2). Total ATP yield from glucose is 38 moles/mole.

Describe citric acid cycle regulation
- most important factor determining flux through the citric acid pathway is the intramitochondrial NAD+/NADH ratio
- increases in acetyl-CoA and succinyl-CoA can reduce cycle flux as well
Pyruvate dehydrogenase-
- controlled by allosteric inhibition and by covalent modification, both governed by the energy state of the cell
- acetyl-CoA, NADH and ATP are allosteric inhibitors of the enzyme; CoA-SH, NAD and AMP are activators
- pyruvate decarboxylase component of the complex is also regulated by a phosphorylation/dephosphorylation cycle (active form is dephosphorylated)
Isocitrate dehydrogenase-
- activated by increase in ADP levels
- low levels of NAD+ can limit the flux through the pathway
What are the anaplerotic (intermediate replenishing) processes for the TCA cycle?
Oxaloacetate
- Carboxylation of pyruvate (animals) by pyruvate carboxylase needing biotin and acetyl-CoA
- Carboxylation of phosphoenolpyruvate (PEP) in plants and bacteria, mediated by PEP carboxylase
Malate
- From pyruvate by malate dehydrogenase using NADPH as cofactor
Reversible transamination Reactions of free amino acids
- Aspartate to oxaloacetate
- Glutamate to alpha-ketoglutarate
What is the glyoxylate cycle?
Animal cells cannot perform net synthesis of carbohydrate from fat.
In plants and some microbes this can be accomplished via the glyoxylate cycle, an anabolic variant of the TCA cycle.
Occurs in specialized organelle, glyoxysome. There is net synthesis of oxaloacetate from acetyl-CoA.
Isocitrate lyase cataylses the conversion of isocitrate to succinate. Unlike the TCA cycle, there is no loss of CO2 in this reaction.
Glyoxysomes are found in seeds of plants.
Plants store energy in the seed as fat.
As the seed germinates, the fatty acids are oxidized to generate acetyl-CoA and this is used to generate glucose for plant growth. In the glyoxysome the acetyl-CoA condenses with oxaloacetate to form isocitrate.
The enzyme isocitrate lyase generates succinate and glyoxylate from isocitrate.
Succinate can then enter mitochondrion for the remaining steps of citric acid cycle in conversion to oxaloacetate.
The glyoxysome also contains malate synthase and and malate dehydrogenase which can generate malate and oxaloacetate. Glyoxylate is converted to malate using acetyl-CoA.
The role of the cycle is to generate succinate for gluconeogenesis using acetyl-CoA from fat oxidation.
No CO2 is lost in the conversion of isocitrate to oxaloacetate in the glyoxysome.
