TCA Cycle Flashcards
Location of TCA
Mitochondria
Products of TCA
NADH, FADH2, GTP, and CO2
Why isn’t there an increase in number of Carbons in this cycle?
The 2 carbons donated by Acetyl-coA will be lost as CO2 from oxaloacetate portion in two reactions of the pathway to compensate.
Even FAs can be transformed into Acetyl-coA …
They can be used to derive energy but not Produce glucose
Info about first step of TCA cycle
1- Citrate (6 carbon sugar) is formed.
2- Enzyme is Citrate Synthase
3- Irreversible
4- Inhibited by high ATP levels
Citrate is an indicator of …?
ATP levels in cell
Regulation of pathways via citrate
1- High levels signal High ATP levels inhibiting glycolysis through PFK1
2- Activates Acetyl-coA Carboxylase (FA synthesis Pathway)
What happens in fasting state to TCA cycle?
Oxaloacetate will be recruited for gluconeogenesis and this decreases the availability of oxaloacetate for Citrate Synthase pulling less Acetyl-coA into the cycle. This leads to A-coA accumulation and activation of Ketone bodies formation.
Information about aconitase
1- Catalyzes the second step in TCA
2- Citrate is the isomer of isocitrate and the intermediate is cis aconitate
3- Reversible
4- Could be inhibited by rat poison fluoroacetate
Isocitrate dehydrogenase
1- Rate limiting step: irreversible 2- Releases CO2 3- Activated by ADP and Ca2+ in skeletal muscles 4- Inhibited by ATP and NADH 5- Releases NADH
Step 4 in TCA cycle: enzyme, activation, inhibition
1- enzyme: alpha ketoglutarate dehydrogenase
2- RDS: irreversible
3- NADH and CO2 release
4- Activation: Ca2+ in skeletal muscles
5- Inhibition: Succinyl-coA high levels and NADH
How is alpha ketoglutarate dehydrogenase complex similar to Pyruvate Dehydrogenase Complex?
1- 3 enzymes work together in the complex
2- Cofactors needed: thiamine, NAD+, FADH, coA, lipoic acid
3- Catalyze irreversible processes
Which step TCA cycle releases energy?
succinyl-coA to succinate releases GTP by
succinyl-coA synthase
Why is the reaction changing succinate to fumarate unique ?
The enzyme succinate dehydrogenase is embedded in the mitochondrial membrane and forms complex II of the ETC. As succinate is changed into fumarate, FADH is reduced to FADH2
What pathways produce fumarate?
1- TCA cycle
2- Urea Cycle
3- Purine synthesis (formation of IMP)
4- Amino acid breakdown: phenylalanine and tyrosine
What is the step of malate into oxaloacetate used for?
1- Provides NADH for the ETC
2- Malate shuttle to transport molecules from cytosol and mitochondria and vice versa:
Malate can cross membrane so acts as a transporter
Moves NADH into the mitochondria and oxaloacetate to cytosol to be used for gluconeogenesis.
TCA intermediates entering other pathways
1- citrate: fatty acid synthesis
2- Alpha keto glutarate: AA Synthesis
3- Oxaloacetate: AA synthesis and Gluconeogenesis
TCA intermediates entering other pathways
1- citrate: fatty acid synthesis
2- Alpha keto glutarate: AA Synthesis
3- Oxaloacetate: AA synthesis and Gluconeogenesis
4- Succinyl-coA: heme synthesis
Where can succinyl-coA come from other than TCA cycle?
Methylmalonyl-coA from Odd chain FAs or Branched AAs breakdown
What are the activators of TCA?
ADP & Ca2+
What are the inhibitors of TCA?
ATP, Acetyl-coA, succinyl-coA, NADH, and citrate
Why is the TCA called amphibolic?
It links anabolic and catabolic pathways.
Define Cellular Energy
Cellular respiration is a set of metabolic reactions that occur in the cell to convert chemical energy from nutrients into ATP.
Anabolism
Synthesis of macromolecules, requires energy, reduction reactions, produces NADPH
Catabolism
Breakdown of macromolecules, generates ATP, oxidation reactions, generated NADH and FADH2
What single intermediate are all proteins, carbohydrates and fatty acids converted into?
Acetyl-coA because it’s the only substrate that could be metabolized by TCA cycle.
What are the 3 Phases of metabolism and where are they located?
1- Cytoplasm: breakdown of macromolecules into their monomers
2- Mitochondria: Monomers are metabolized to produce TCA cycle intermediates, acetoacetyl-coA, pyruvic acid, and Acetyl-coA
3- Mitochondria: TCA cycle and ETC oxidative phosphorylation
What will the metabolism of amino acids release other than the usual products?
NH3 which will be dealt with in urea cycle.
What could each macromolecule contribute to in TCA ?
AAs: oxaloacetate or pyruvate or Acetyl-coA
FAs: Acetyl-coA
Glucose: Pyruvate
Aconitase activity?
Keq favors Citrate and not Isocitrate. However, more Isocitrate is formed because of the next in line irreversible reaction that consumes Isocitrate. (Le Chatelier Principle).
- Citrate: non-oxidizable 3⁰ alcohol; Isocitrate: oxidizable 2⁰ alcohol; more reactive.
- If Citrate levels are high, this favors Fatty Acid synthesis.
Aconitase can distinguish between the upper (CH2COO-) added by Acetyl-CoA in Citrate, and the lower (CH2COO-) which belongs to Oxaloacetate. => The dehydration then rehydration occur between the lower (CH2COOH) from the OXAC backbone and (-OH) => Aconitase considers the Citric Acid molecule as Prochiral. Although Citrate is a symmetrical molecule (achiral; with a plane of symmetry), Aconitase reacts with it asymmetrically so that the 2 carbon atoms that are lost in subsequent reactions of the cycle are not those that were added by Acetyl CoA (i.e. we eventually lose 2 carbons from the Oxaloacetate portion, and not the acetyl CoA portion).
=> Aconitase catalyzes dehydration – rehydration of Citrate, by modifying the Oxaloacetate Portion ONLY, which changes the Symmetrical Citrate into Asymmetrical Isocitrate.
How many isozymes does Isocitrate dehydrogenase have and do they all function in TCA?
3 isozymes. 1 in mitochondria uses NAD+ in TCA
2 in mitochondria and cytosol used NADP+ outside the scope of TCA.
What chemically happens in the step of transforming isocitrate to alpha keto glutarate?
Chemically, the rxn entails: oxidation of the 2⁰ alcohol into keto, and decarboxylation of the central (COOH).
The alpha keto glutarate dehydrogenase is similar to what enzyme?
It is a multienzyme complex similar to PD. It performs 3 steps: decarboxylation results in acetaldehyde which is then oxidized into acetic acid and esterified thru thioester bond. Both multienzymes require the following cofactors: TPP (thiamine pyrophosphate), NAD+, FADH, and coA
What is so special about thioester hydrolysis?
Produces enough energy to drive substrate level phosphorylation such as changing GDP to GTP in succinyl-coA to succinate step.
How many isoenzymes does Succinyl-coA synthetase have?
2: in gluconeogenic tissues both are present (specific to ADP and GDP). In nongluconeogenic, only the one specific to ADP is present.
When reactants are regenerated in products what does that mean?
Intermediates are acting catalytically.
Succinate dehydrogenase
Same as complex II in ETC. It is embedded in inner mitochondrial membrane not like the rest in the matrix. Requires FADH. (whenever there is an alkene, FADH is required).
The overall reaction of TCA ? How much ATP does it generate?
Per Acetyl Co-A:
3 NADH 9 ATP (Oxidative phosphorylation)
1 FADH2 2 ATP (Oxidative phosphorylation)
1 GTP 1 ATP (Substrate level phosphorylation)
12 ATP / Acetyl Co-A = 24 ATP / glucose molecule (2 Acetyl-CoA / glucose molecule)
What explains the fact that FAs can’t be transformed into glucose?
The pyruvate to Acetyl-coA is irreversible.
What are the 3 enzymes that have similar mechanisms?
Oxidative decarboxylation enzymes:
Pyruvate dehydrogenase
Alpha keto glutarate dehydrogenase
BCAA; Branched chain amino acyl keto dehydrogenase
Location of PD?
Mitochondrial matrix
PD multienzyme complex enzymes, core enzyme, their names, and functions.
PD is a cluster of 3 main enzymes (E1, E2, E3), in addition to other enzymes. Each one of the PD enzymes exists in multiple copies, the number and size of which varies among organs and tissues.
- The core protein of the PD enzyme complex is E2 (exists as many as 60 copies) to which E1 and E3 are attached:
• E1 = Pyruvate Dehydrogenase (old name):
- E1 is linked to TPP.
- E1 has a Decarboxylase Activity.
• E2 = Dihydrolipoyl Transacetylase:
- E2 is covalently linked to a Disulfide Lipoic Acid, via a Lysine residue.
- 2 regulatory proteins are part of E2 as well: a protein kinase and a phospho-protein phosphatase.
• E3 = Dihydrolipoyl Dehydrogenase:
- E3 is linked to FAD (oxidized form)
The process of oxidative Decarboxylation at the PD Complex:
1. At E1, Pyruvate gets decarboxylated (CO2 is lost), and the product is linked at the aldehydic level to TPP (cofactor of E1) .
2. The aldehyde gets oxidized by E2, at the expense of reducing the (S-S) of lipoic acid bound to E2 into (-SH).
- We then have a Thio-esterification rxn, where one reduced (-SH) acts like an (-OH) attacking the (C=O) of the aldehyde. We now have an acetyl linked to the S of lipoic acid.
(In Ester, we have an -O attached to (C=O); In Thio-ester, we have an -S attached to (C=O))
=> 2 enzymatic rxns happen at the level of E2: Oxidation and Thio-esterification.
3. Transesterification with CoA-SH occurs, releasing acetyl CoA in the matrix of the mitochondria, leaving behind reduced lipoyllysine.
4. The FAD linked to E3 oxidizes Lipoyl group on E2. => FAD on E3 is reduced to FADH2.
5. The last reaction is re-oxidation of FADH2 by NAD+ cofactor to regenerate the active enzyme with: E1-TPP, E2 lipoic (oxidized) and E3 FAD (oxidized) linked.
- The ultimate result is Pyruvate going into acetyl CoA, while releasing NADH. => This reaction generates approximately 3 ATP from NADH.
- It is true FAD was reduced, but it was then re-oxidized, and it is not released into the matrix and doesn’t feed into the ETC. => FAD will not contribute to ATP.
What are the 7 ways TCA is regulated?
TCA is regulated mainly at the PD rxn level but also at other levels as well:
1. Product Feedback Inhibition (PD):
On the PD, NADH competes with the NAD binding site, and acetyl CoA competes with the CoA binding site.
- Thus, ↑ NADH or ↑Acetyl CoA => Competitive Inhibition of PD (Feedback Inhibition)
- Allosteric Inhibition (PD):
- NADH is also an Allosteric Inhibitor of E3 on PD.
- Acetyl CoA is also an Allosteric Inhibitor of E2 on PD. - Covalent Modification (PD) – Hormone Triggered:
- E2 of PD has a Phosphatase and a kinase activity.
- PD can be phosphorylated at E1 (Ser).
- PD is phosphorylated: Inactive; PD is dephosphorylated: Active.
- As ↑ NADH or ↑Acetyl CoA or ↑ ATP => Positive Modulators of the kinase activity of E2 => PD is phosphorylated => PD is Inactive.
- Insulin Effect (Activates PD):
- Insulin activates the phosphatase activity of E2 => PD is dephosphorylated => PD is Active. - Ca2+ Effect (Activates PD):
- As ↑ Ca2+ (due to increased muscular contraction) => Ca2+ activates the phosphatase activity of E2 => PD is dephosphorylated => PD is Active. (Glycolysis is also stimulated to break down Glycogen into Glucose, and then generate Pyruvate)
- As ↑ Ca2+ => ICDH is activated, and alpha-keto glutarate dehydrogenase is activated.
- ETC:
- The activity of TCA depends on the continuous supply of oxidized NAD and FAD through the ETC. - ADP (Activates TCA):
- As ↑ ADP => TCA and ETC are favored; ADP is a substrate for ETC:
If ↓ ADP => ↓ rate of ETC => ↓ NAD and FAD => ↓ rate of TCA.
- ADP is an allosteric activator of ICDH; ADP increases the affinity (↓ Km) of ICDH towards Isocitrate.
State other effectors of the TCA.
1- Arsenicals (poisonous) compounds specifically interact irreversibly with thiol of lipoic acid.
2- Thiamine deficiency is characterized by inability to metabolize Pyruvate via PD. Pyruvate piles up, then it gets reduced by Lactate Dehydrogenase (LDH) into Lactate.
=> A ↓ Pyruvate/Lactate indicates a problem with aerobic metabolism, leading to Lactic Acidosis.
- Note that Lactic Acid will increase in case PD has any mutation in any of its subunits, or if there is problem in any of the ETC complexes. In general, anything that affects aerobic metabolism is characterized by Lactic Acidosis.
- Other affected enzymes by Thiamine Deficiency are those having catalytic cycle similar to PD, such as alpha keto glutarate dehydrogenase.
- Brain is quite affected by Thiamine deficiency because it utilizes mainly glucose generating Pyruvate. Alcoholics excrete Thiamine, so may present with symptoms of increasing lactic acid levels.Malonic acid is a natural reversible competitive inhibitor Succinate dehydrogenase.
Maleate is the cis-isomer of fumarate leading to a reversible competitive inhibition of fumarase.
Fluoroacetate reacts with OXAC, yielding Fluorocitrate that inhibits Aconitase.Arsenicals (poisonous) compounds specifically interact irreversibly with thiol of lipoic acid.Thiamine deficiency is characterized by inability to metabolize Pyruvate via PD. Pyruvate piles up, then it gets reduced by Lactate Dehydrogenase (LDH) into Lactate.
=> A ↓ Pyruvate/Lactate indicates a problem with aerobic metabolism, leading to Lactic Acidosis. - Note that Lactic Acid will increase in case PD has any mutation in any of its subunits, or if there is problem in any of the ETC complexes. In general, anything that affects aerobic metabolism is characterized by Lactic Acidosis.
- Other affected enzymes by Thiamine Deficiency are those having catalytic cycle similar to PD, such as alpha keto glutarate dehydrogenase.
- Brain is quite affected by Thiamine deficiency because it utilizes mainly glucose generating Pyruvate. Alcoholics excrete Thiamine, so may present with symptoms of increasing lactic acid levels.Malonic acid is a natural reversible competitive inhibitor Succinate dehydrogenase.
Maleate is the cis-isomer of fumarate leading to a reversible competitive inhibition of fumarase.
Fluoroacetate reacts with OXAC, yielding Fluorocitrate that inhibits Aconitase.
