1. 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
  2. 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)
  3. 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
  4. 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
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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
  10. 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
  11. 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!
  12. 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+
  13. 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)
  14. 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
  15. 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
  16. 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)
  17. 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)
  18. 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
  19. 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)
  20. 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?
  21. 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
Card Set
Metabolism- TCA cylce/Krebs cycle/Citric acid cycle