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Citric acid cycle (overview)
- pricipal gateway to aerobic metabolism
- ***important source of building blocks for AA, glucose, nuleotide bases, cholesterol and porphyrin (organic part of heme)
- it is the condensation of a 40carbon molecule (oxaloacetate) with a 2-Carbon acetyl unit (acetyl CoA) to yield a 6-C tricarboxlic acid (citrate) which is sequentially decarboxylated and oxidised to regenerate oxaloacetate
- overall function to harvest high energy electrons from carbon fuels
- net yield/cycle: 2CO2 + 3NADH + 1 FADH2 (note: no ATP made or O2 needed)
- the 8e- carried by NADH and FADH2 are destined for oxidative phosphorylation
- oxidative phosphorylation 90-95% of ATP formed
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Functions of TCA cycle
- Energy provision:
- energy from carbohydrate, fat & protein
- final oxidation of Carbon compounds to CO2 and H2O
- reduced cofactors (NADH and FADH2) for oxidative phosphorylation
- substrate for biosynthesis of macromolecules:
- substrate for glucose synthesis
- substrate for amino acid/protein
- substrate for porphyrin and haem (haem comprises porphyrin and iron)
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Links between glycolysis and citric acid cycle
- glycolysis ends w/ formation of pyruvate in the cytoplasm
- pyruvate transported to the mitochondrial matrix where it is oxidised and decarboxylated by the pyruvate dehydrogenase complex (PDH) to form acetyl CoA
- Net rxn: Pyruvate + NAD+ + CoA --> acetyl CoA + CO2 and NADH
- conversion of pyruvate to acetyl CoA is an irreversible rxn, with capture of high transfer potential electrons to NADH
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Pyruvate dehydrogenase complex
- Giant complex with 3 different enzymes (pyruvate dehydrogenase, dihydrolipoyl transacetylase and dihydrolipoyl dehydrogenase) each enzyme consists of several polypeptides and catalytic cofactors (eg thiamine pyrophosphate, lipoamide and FAD)
- **Thiamine deficiency leads to beriberi: symptoms related to nerve damage associated with malfunction of PDH and a-ketoglutarate dehydrogenase complexes
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Substrates for TCA
- Acetyl coenzyme A:
- from pyruvate and glycolysis
- from fatty acid breakdown (B-oxidation)
- from ketone bodies (liver during starvation)
- from ketogenic amino acids
- other carbon sources:
- a-ketoglutarate from amino acid breakdown
- succinyl CoA from propionate (volatile fatty acid- very important in ruminants!!)
- oxidised cofactors:
- NAD+ and FAD (TCA operates under aerobic conditions b/c these electron acceptors are regenerated when NADH and FADH2 transfer e- to O2 in e-transport chain
- GDP and Pi
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Products of TCA cycle
- 2 x CO2 --> released in breath
- 3 x NADH --> ox phos (3 ATP)
- 1 x FADH2 --> to ox phos (2 ATP)
- GTP
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Location of TCA cycle
- Tissue location:
- all tissues w/ mitochondria
- not in RBC
- Intracellular location:
- in mitochondria
- in matrix of mitochondria
- Mitochondrial origin:
- synergistic bacteria
- has it's own DNA
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Physiology of the TCA cycle
- Up-regulation
- increased demand for energy
- exercise: aerobic when oxygen supply is balanced to demand
- anaerobic when oxygen supply can not meet demand
- temperature decrease in environment
- disease
- Down-regulation:
- rest, hibernation, recovery
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Control of TCA cycle
- Whole body control:
- Endocrine and CNS control, exercise increases
- environmental effectors, fight or flight
- cellular control:
- energy levels (ATP/ADP (AMP) ratios, NADH/NAD+ ratios)
- regulatory enzymes responding to metabolites
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Pathophysiology of TCA and Ox Phos
- few diseases affect TCA/Ox Phos
- if distrupted it's usually fatal
- Toxins/poisons can inhibit TCA/Ox Phos
- Cyanide, Rotenone, Oligomycin, Bromethalin
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Features of the TCA cycle
- 4 Carbon acid (oxaloacetate) combines w/ 2 carbon unit acetly group of acetyl CoA
- over further 7 reactions 2 x CO2 released
- this is a net oxidation of the carbon as in burning wood
- released energy is kept under control, converted to useful energy (ATP) not heat & light
- oxaloacetate is restored at end of the 7 reactions!
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BC: before cycle
- irreversible funneling of pyruvate (product of glycolysis) into TCA cycle
- pyruvate can cross inner mitochondria membrane
- converted by pyruvate dehydrogenase, a multi enzyme complex that requires 5 co-enzymes (thiamine pyrophosphate, lipoic acid, CoA, FAD, NAD+)
- 3 reaction process
- CO2 released
- NAD+ reduced to NADH and H+
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Steps in citric acid cycle
- 1) condensation of oxaloacetate (C4) and acetyl CoA (C2) to 6-carbon citrate, catalysed by citrate synthase
- 2) citrate is isomerized to isocitrate by aconitase, an iron-sulphur enzyme
- 3) isocitrate is oxidised and decarboxylated to a-ketoglutarate by isocitrate dehydrogenase. the 1st pair of high energy electrons formed (NADH)
- - Isocitrate dehydrogenase inhibited by: ATP and NADH when cell is energy rich leads to: excess isocitrate, excess citrate (substrate for fatty acid synthesis)
- 4) a-ketoglutarate is also oxidised and decarboxylated to succinyl CoA, mediated by the a-ketoglutarate dehydrogenase complex (homologous to the PDH complex) succinyl CoA formed with a 2nd NADH
- 5) cleavage of thioester bond of succinyl CoA is coupled to phosphorylation of GDP to form GTP and succinate, catalysed by succinyl CoA synthase
- 6) FINALLY, succinate is regenerated to oxaloacetate in 3 steps: oxidation, hydration, and a 2nd oxidation (energy yield from these 3 steps: FADH2 and a 3rd NADH)
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Succinate dehydrogenase
- membrane bound on inner mitochondrial membrane
- domain in matrix to react w/ succinate
- contains FAD as cofactor (b/c free E change insufficient to reduce NAD+)
- trans double bond formed i fumarate
- FADH2 enters electron trandsport chain & oxidative posphorylation
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Control of Pyruvate dehydrogenase complex
- in animals, formation of acetyl CoA from pyruvate is key irreversible step b/c they are unable to convert Acetyl CoA into glucose
- oxidative decarboyxlation of pyruvate to acetyl CoA commits carbon atoms of glucose to 2 fates:
- 1) oxidation to CO2 by TCA cycle (& generation of E)
- or
- 2) incorporation into lipid
- PDH complex at critical decision point in metabolism (tightly regulated)
- inhibited by products of PDH (high levels of Acetyl CoA and NADH. CoA and NAD+ reverse the inhibitory effects)
- controlled by E charge. PDH component (of the complex) is inhibited by GTP and activated by ADP
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Regulation by reversible phosphorylation (TCA cycle- pyruvate dehydrogenase complex)
- 1) inactive when serines are phosphorylated (by PDH kinase family members)
- 2) Active when serives are dephosphorylated (by PDH phosphatase family members)
- 3) insulin stimulates dephosphorylation (accelerates pyruvate to Acetyl CoA and hence glucose to pyruvate)
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Rate adjusted to meet cell's need for ATP (TCA)
- ATP is allosteric inhibitor of citrate synthase
- ADP allosterically stimulates isocitrate dehydrogenase
- high E charge inhibits a-ketoglutarate dehydrogenase
- Activity of citrate synthase (ATP is allosteric inhibitor- increases Km for acetyl CoA)
- Activity of isocitrate dehydrogenase (inhibited by NADH- displaces NAD+, and ATP) (activated by NAD+- enhances affinity for substrates and ADP)
- Activity of a-ketoglutarate dehydrogenase (inhibited by succinyl CoA, NADH (products of the reaction it catalyses) and ATP (high energy Charge)
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Energy yield comparison
- Net stoichiometry from electron transport chain (ox phos) is about 3 ATP per NADH, and 2 ATP per FADH2
- 1 round citric acid cycle produces 1 GTP, 3 NADH and 1 FADH2, equivalent to about 12.0 ATP, 1 acetyl unit generates 12 ATP. also 1 NADH from pyruvate to acetyl CoA conversion. hence total of 15 ATP from pyruvate to water
- in glycolysis, 1 glucose molecule generates 2 acetyl CoA and a net of 2 ATP
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Maintaining the TCA cycle
- TCA intermediates can be used for biosynthesis
- Net loss of substrate reduces rate of cycle and ATP production
- replenished by 'anaplerotic reactions' an enzyme catalysed chemical rxn that recharges the supply of intermediate molecules
- oxaloacetate is used for gluconeogenesis especially in ruminants and carnivores
- pyruvate is converted to oxaloacetate by pyruvate carboxylase reaction (see GNG)
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Lactate acidosis: patients in shock will often suffer from lactate acidosis due to deficiency of oxygen (circulatory collapse)
1) why does a lack of oxygen lead to lactic acid build-up?
2) besides oxygen, would you advocate the use of dichloracetate (inhibitor of the kinase associated w/pyruvate dehydrogenase) and why for this tx?
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The IV infusion of fructose into healthy volunteers leads to a 2- to 5- fold increase in lactate levels in the blood. a far greater increase than that observed after the infusion of the same amount of glucose
1) speculate why lactate accumulation was greater w/ fructose infusion
2) explain if the use of intravenous fructose is a good idea
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