Tricarboxylic Acid (TCA) Cycle
- most fuels ultimately enter the TCA cycle when O\(_2\) is present
- location: mitochondrial matrix
Mitochondrial structure (relevant features)
Outer membrane
- contains porins: small molecules and ions can move in/out
- intermembrane space composition is similar to cytoplasm
Inner membrane
- high protein:lipid ratio (ETC proteins)
- contains many TIMs (translocase of inner membranes)
- cristae = folds of inner membrane that increase surface area
Matrix
- enzyme-rich compartment where TCA enzymes operate
Step 0: Pyruvate oxidation (link reaction)
Reaction:
- pyruvate + CoA-SH → acetyl-CoA + CO\(_2\)
Notes:
- acetyl group is linked to the -SH group of CoA
- catalyzed by pyruvate dehydrogenase complex (PDC)
- pyruvate is transported into the matrix via TIMs
Pyruvate dehydrogenase complex (PDC)
Enzymes and coenzymes :
1) Pyruvate dehydrogenase (E1)
Coenzyme:
- thiamine pyrophosphate (TPP), vitamin B\(_1\)
Function:
- converts pyruvate + TPP → hydroxyethyl-TPP + CO\(_2\)
2) Dihydrolipoyl transacetylase (E2)
Coenzymes:
- lipoamide
- CoA (vitamin B\(_5\))
Functions:
- hydroxyethyl-TPP + lipoamide → TPP + acetyl-dihydrolipoamide
- acetyl-dihydrolipoamide + CoA → acetyl-CoA + fully reduced lipoamide
(then re-oxidation coupling)
- fully reduced lipoamide + FAD → FADH\(_2\) + lipoamide (via dihydrolipoyl dehydrogenase)
3) Dihydrolipoyl dehydrogenase (E3)
Coenzymes:
- FAD
- NAD\(^+\)
Function:
- FADH\(_2\) + NAD\(^+\) → FAD + NADH
Regulation of PDC
PDC kinase
- phosphorylates PDC and inactivates it
Allosteric regulation :
- up: acetyl-CoA, NADH
- down: ADP, pyruvate
PDC phosphatase
- removes phosphate and activates PDC
Allosteric regulation :
- up: Ca\(^{2+}\)
Product inhibition
- NADH and acetyl-CoA inhibit the complex
The TCA cycle (9-step listing)
1) Citrate synthase
- acetyl-CoA + oxaloacetate → citrate + CoA-SH (CoA-SH leaves in exchange for water)
Notes:
- product inhibition (citrate binds active site)
- citrate slows PFK-1
2) Aconitase
- citrate ⇌ cis-aconitate (dehydration)
- cis-aconitate ⇌ isocitrate (hydration)
3) Isocitrate dehydrogenase
- isocitrate → oxalosuccinate → \(\alpha\)-ketoglutarate + CO\(_2\)
- NAD\(^+\) → NADH
- requires proton
Regulation :
- up: ADP, NAD\(^+\)
- down: ATP, NADH
4) \(\alpha\)-Ketoglutarate dehydrogenase complex
- \(\alpha\)-ketoglutarate + CoA-SH + NAD\(^+\) → succinyl-CoA + CO\(_2\) + NADH
Notes :
- product inhibition by succinyl-CoA and NADH
-
has 3 enzymes:
-
\(\alpha\)-ketoglutarate dehydrogenase
- dihydrolipoyl succinylase
- dihydrolipoyl dehydrogenase
- uses the same coenzymes as PDC
5) Succinate thiokinase
- succinyl-CoA + P\(_i\) + GDP → succinate + CoA-SH + GTP
6) Succinate dehydrogenase (membrane-associated)
- succinate + FAD → fumarate + FADH\(_2\)
7) Fumarase
- fumarate + OH\(^-\) → carbanion transition state → malate (with H\(^+\))
8) Malate dehydrogenase
- malate + NAD\(^+\) → oxaloacetate + NADH
Note :
- this is effectively driven forward by coupling to citrate synthase (acetyl-CoA + oxaloacetate is highly exergonic)
Energy output (no ETC accounting beyond NADH/FADH\(_2\) ATP equivalents)
Assume:
- NADH = 2.5 ATP
- FADH\(_2\) = 1.5 ATP
Glycolysis (stage 1)
- 2 NADH, 2 pyruvate, 2 ATP
- total: 7 ATP equivalents
Pyruvate oxidation (stage 2)
- 2 NADH
- total: 5 ATP equivalents
Citric acid cycle (stage 3)
- 6 NADH, 2 FADH\(_2\), 2 GTP
- total: 20 ATP equivalents
Anaerobic respiration
- 2 ATP
Anaplerotic reaction (refilling TCA intermediates)
- pyruvate + HCO\(_3^-\) + ATP → oxaloacetate enzyme: pyruvate carboxylase
Regulation :
- acetyl-CoA is an allosteric activator
Coenzyme:
- biotin, vitamin B\(_7\)
Inhibitors of TCA / linked steps
Sodium fluoroacetate
- converted to fluorocitrate
- fluorocitrate inhibits aconitase
Arsenite
- binds fully reduced lipoamide
- inhibits PDC and \(\alpha\)-ketoglutarate dehydrogenase complex
Clinical note: PDC deficiency
- insufficient PDC activity → increased lactate production (lactic acidemia)
- may be relieved by high-fat, low-carbohydrate diet