TCA cycle Flashcards
What are the different names of this cycle?
Citric acid cycle, Krebs cycle and Tricarboxylic acid cycle (TCA)
How does pyruvate then move into the TCA cycle?
Pyruvate is converted into Acetyl-CoA in the link reaction
Describe the link reaction?
Pyruvate + CoA + NAD ⇌ Acetly-CoA + CO2 + NADH
Using pyruvate dehydrogenase
Describe the effect of pyruvate dehydrogenase?
It is a multienzyme complex (enzymes non-covalently linked - a metabolon), which is efficient as they are very compact leading to ledd diffusion space between releasing a product to be further catalysed
They minimise side reactions and have a more control over the reaction
Describe the compostion of pyruvate dehydrogenase?
It is composed of 3 enzymes: E1 - pyruvate dehydrogenase E2 - dihydrolipoyl transacetylase E3 - dihydrolipo dehydrogenase 24 E2 forms a cube, surrounded by 24 E1, whihc is then surrounded by 12 E3 This forms a large cube 10 mega Da It requires 5 cofactors
What are the cofactors of pyruvate dehydrogenase?
Thiamine Lipoic acid Coenzyme A (CoA) Flavin adenine dinucleotide (FAD) Nicotinamide adenine dinucleotide (NAD+)
How does pyruvate dehydrogenase work? Step 1 and 2
- Pyruvate dehydrogenase (E1), and TPP, decarboxylates pyruvate with the formation of a hydroxyethyl‐TPP intermediate
- The hydroxyethyl group is transferred to the next enzyme, dihydrolipoyl transacetylase (E2) (containing a lipoamide group),
The hydroxyethyl attacks the lipoamide disulfide, and TPP is eliminated = E1 regenerated and produces Acetyl-dihydrolipoamide-E2
How does pyruvate dehydrogenase work? Step 3, 4 and 5
- E2 then catalyzes a transesterification reaction in which the acetyl group is transferred to CoA, producing acetyl‐CoA and dihydrolipoamide‐E2
- Dihydrolipoyl dehydrogenase (E3) reoxidizes dihydro‐lipoamide to complete the catalytic cycle of E2, E3 is now reduced
- Finally, reduced E3 is reoxidized. The sulfhydryl groups are reoxidized by a mechanism in which FAD funnels electrons to NAD+, yielding NADH
What does E2 contain and how does it work?
E2 contains a lipoic acid and a lysine attached - which is known as the swinging arm
It moves and visits all the different active sites in the E1, E2 and E3 enzymes
The distance between the centre of the arm and where it needs to reach in the active site isn’t long enough (longer than 14 A)
E2 actually has a disorganised section and then a domain which contains the lipoic acid
The disorganised section can also move - which makes the reach of the arm much longer than it seems
The arm isn’t completely flexible but it can articulate
What is step 1 of the TCA cycle?
Using citrate synthase catalyses the condensation of oxaloacetate (OAA) to acetyl-CoA to form citrate (releasing CoA)
Step 1 of the TCA cycle - describe citrate synthase?
A homodimer with 2 domains forming a cleft containing the substrate binding site
It has an open binding site, and when the substrate binds to it the smaller domain undergoes a 18 degree rotation which closes the cleft
Step 1 of the TCA cycle - what is the mechanism of citrate synthase?
- Rate limiting formation of acetyl-CoA enolate, stablised by a hydorgen bond from His 274
- Nucleophilic attack of acetly-CoA enolate on oxaloacetate’s carbonyl carbon to produce citryl-CoA
- Citryl-CoA hydrolysis: forming citrate and CoA.
This step produces ∆G = -31.5 kJ mol^-1
What is step 2 of the TCA cycle?
Citrate is converted into it’s isomer isocitrate using aconitase
This forms a cis-aconitate intermediate
Step 2 of the TCA cycle - describe aconitase?
It contains a [4Fe-4S] iron-sulfur cluster
This coordinates the OH group of citrate to facilitate its elimination
Aconitase is multifunctional and is also involved in the regulation of iron homeostasis
Step 2 of the TCA cycle - what is the mechanism of aconitase?
- Dehydration, where H2O is removed (citrate is prochiral, therefore H2O is only removed from citrates lower arm
- The intermediate cis-aconitate is rehydrated to remove the double bond - which could potentially yield 4 stereoisomers
However, aconitase catalyzes the stereospecific addition of OH− and H+ to produce only one isocitrate stereoisomer
What is Step 3 of the TCA cycle?
Isocitrate dehydrogenase catalyses oxidative decarboxylation from isocitrate into a-ketoglutarate (secondary alcohol to a ketone)
It forms a transient intermediate - oxalosuccinte and releases NADH and CO2
Step 3 of the TCA cycle - describe Isocitrate dehydrogenase?
It is NAD+ dependent and also requires a Mn2+ or Mg2+ cofactor
Step 3 of the TCA cycle - describe this stage?
- NAD+ dependent isocitrate deydrogenase catalyses isocitrate to oxalosuccinate
- Decarboxylation of oxalosuccinate to a ketone
(Mn2+ helps polarize the newly formed carbonyl group) - A proton is added to form a-ketoglutarate
What is step 4 of the TCA cycle?
a-ketoglutarate dehydrogenase catalyses oxidative decarboxylation from a-ketoglutarate to succinyl-CoA
CoASH and NAD+ are needed to produce CO2 and NADH
Step 4 of the TCA cycle - describe a-ketoglutarate dehydrogenase?
Similar to pyruvate dehydrogenase as it is a multienzyme complex:
E1 - a-keto glutarate dehydrognase
E2 - Dihydrolipoyl transsuccinylase
E3 - Dihydrolipoyl dehydrogenase
Uses 5 cofactors and produces a high energy ‘thioester’ succinyl-CoA
What is step 5 of the TCA cycle?
Succinyl-CoA synthetase (or succinate thiokinase) catalyses the cleavage of succinyl-CoA into succinate
Producing a high energy molecule:
Animals - GTP from GDP +Pi
Plants and bacteria - ATP from ADP + Pi
Step 5 of the TCA cycle - what is the mechanism of succinyl-CoA synthetase?
- Succinyl‐CoA reacts with Pi to form succinyl‐phosphate (high energy) and CoA.
- The phosphoryl group is then transferred from succinyl‐phosphate to a His residue on the enzyme (3-phospho-His), releasing succinate.
- The phosphoryl group on the enzyme is transferred to GDP, forming GTP
There are high energy intermediates all the way through that we want to move through very quickly!
This is substrate level phosphorylation
What is step 6 of the TCA cycle?
Succinate dehydrogenase catalyses the stereospecific dehydrogenation of succinate to fumarate
This produces FADH2 from FAD linked to the enzyme
Step 6 of the TCA cycle - describe the enzyme succinate dehydrogenase?
It contains a FAD prosthetic group covalently linked to the enzyme via a His residue
It is the only membrane‐bound enzyme of the citric acid cycle so it is positioned to funnel electrons directly into the electron transport machinery of the mitochondrial membrane
Step 6 of the TCA cycle - what is an inhibitor associated with this step?
Malonate is a competitive inhibitor to succinate
This is how Krebs confirmed this pathway was a cycle
What is step 7 of the TCA cycle?
Fumarase catalyses the hydration of the double bond of fumarate to malate
This proceeds via a carbanion transition state
The OH- addition occurs before the H+ addition
What is step 8 of the TCA cycle?
Malate dehydrogenase catalyses an oxidation reaction of malate into regenerated oxaloacetate
Step 8 of the TCA cycle - how does the mechanism of malate dehydrogenase work?
NAD+ transfers a hydride ion, resulting in the hydroxyl group of malate being oxidised
Step 8 of the TCA cycle - what are the thermodynamics of this stage?
ΔG = +29.7 kJ mol^-1
Therefore the concentration of oxaloacetate is low at equilibrium
However, citrate synthetase was ΔG = -31.5 kJ mol^-1, which allows citrate formation to be exergonic even at the low oxaloacetate concentrations present in cells and therefore helps keep the TCA cycle moving
What is the overall production of ATP at this point?
(there are two TCA cycles per 1 glucose)
6 NADH -> 15 ATP
2 FADH2 -> 3 ATP
2 GTP -> 2 ATP
Glycolysis + TCA = 32 ATP
What happens at the end of the TCA cycle?
This pathway is located in the mitochondria
The products have to be transported out, just as the reactants had to be transported in
What is a common mistake about the TCA cycle?
The 2 atoms of carbon that we put in are not the 2 carbon atoms released as CO2 - which we could see from the radioactive labelling (tracers)
What is regulated in the TCA cycle?
Pyruvate dehydrogenase The rate determining enzymes: Citrate synthase Isocitrate dehydrogenase a-ketoglutarate dehydrogenase
How is pyruvate dehydrogenase regulated in the TCA cycle?
By product inhibition: byt NADH and acetyl-CoA
Covalent modification by the de/phosphorylation of E1 - pyruavte dehydrogenase kinase can inactivate it
How are the rate determining enzymes controlled?
- Substrate availability
- Product inhibition
- Competitive feedback inhibition by intermediates further along the cycle
- Allosteric actiavtors: ADP and Ca2+
There is no single control point, as flux control is distributed among several enzymes
What type of reactions does the TCA cycle involve?
It is amphibolic - anabolic and catabolic
What other reactions can be linked to the TCA cycle?
Synthesis and degredation of amino acids
Synthesis of fatty acids and cholestrol
Synthesis of glucose
Synthesis of porphyrins
What intermediates can synthesise amino acids?
a-ketoglutarate and oxaloacetate: all
Fumarate: Asp, Phe, Tyr
Succinyl-CoA: Ile, Met, Val (also synthesises porphyrins)
What intermediates synthesis fatty acids and cholestrol?
Citrate
Odd-chain fatty acids can be used to form succinyl-CoA
What intermediate synthesises glucose?
Oxaloacetate is converted into Malate for transport out the mitochondrion
What are types of side reactions to the TCA cycle?
Cataplerotic reactions: reactions that depleted intermediates (If an intermediate is used in a side reaction it can cause the TCA cycle to stop/slow)
Anaplerotic reactions: build up the intermediates (mainly pyruvate and amino acids - some odd-chain fatty acids)
