BIOS 301 - TCA

Stage 1 of Respiration

• Acetyl-CoA production

• Oxidation of fatty acids, glucose, and some amino acids generates NADH to yield acetyl-CoA

• Carbohydrates release 1/3 of total potential CO2 during Stage 1

Stage 2 of Respiration

• Acetyl-CoA oxidation

• Generates 3 NADH, 1 FADH2, and 1 GTP

• Remaining carbon atoms from carbohydrates, amino acids, and fatty acids are released during Stage 2

Stage 3 of Respiration

• Oxidative phosphorylation

• Electrons carried by NADH and FADH2 pass through ETC to generate the vast majority of ATP during catabolism, ultimately reducing O2 to H2O

The Citric Acid Cycle

Synthase

No ATP/GTP involved

Synthetase

ATP/GTP involved

Induced fit in citrate synthase

• OAA and acetyl-CoA bind sequentially

• OAA binding causes conformational change that creates binding pocket for acetyl-CoA

• Avoids unnecessary hydrolysis of thioester in acetyl-CoA

Describe function of aconitase as an iron response regulatory protein

• Low [iron] causes loss of Fe-S cluster from aconitase

• Aconitase then binds mRNA to regulate gene expression:

  • Suppresses translation of ferritin (stores iron)

  • Enhances translation of transferrin receptor (uptakes iron)

Origin of C-atoms in CO2

• All CO2 released during the citric acid cycle is produced before succinyl-CoA is made

• Both CO2 molecules lost were present on the oxaloacetate that entered the cycle (i.e. not directly from glucose)

Reaction series of succinyl-CoA synthetase

• Phosphoryl oxygen of Pi attacks carbonyl of succinyl CoA, releasing CoA-SH

• Histidine N3 attacks phospho-succinyl intermediate, forming P-N bond and releasing succinate

• Pi group is transferred from His to GDP to form GTP

Succinyl-CoA Synthetase catalytic site

• Partial positive charges of the helix dipoles (one each from the alpha and beta subunits) stabilize the phosphohistidyl group

• The alpha subunit is specific for either GDP or ADP and distinguishes the two isozymes

• Not stereospecific with respect to C2-C3 plane of symmetry (as in aconitase reaction)

Alternative function of succinyl dehydrogenase (outside TCA)

• Functions as Complex II of the Electron Transport Chain

• Serves as an integral membrane protein of the inner mitochondrial membrane

General sequence of events in citric acid cycle

• Step 1: C-C bond formation to between acetate (2C) and oxaloacetate (4C) to make citrate (6C)

• Step 2: Isomerization via dehydration/rehydration

• Steps 3-4: Oxidative decarboxylations to give 2 NADH

• Step 5: Substrate level phosphorylation to give 1 GTP

• Step 6: Dehydrogenation to give 1 FADH2

• Step 7: Hydration

• Step 8: Dehydrogenation to give NADH

Points of regulation of Citric Acid Cycle

• Regulated at highly thermodynamically favorable (irreversible) steps

  • Pyruvate dehydrogenase complex

  • Citrate synthase

  • Isocitrate dehydrogenase

  • Alpha-ketoglutarate dehydrogenase complex

TCA general regulatory mechanism

• Activated by substrate availability, inhibited by product accumulation

• Overall products of pathway are NADH and ATP

  • Inhibitors (NADH, ATP)

  • Activators (NAD+, AMP/ADP)

Regulation of pyruvate dehydrogenase complex

• Regulated by reversible phosphorylation of E1

  • Phosphorylation: inactive

  • Dephosphorylation: active

• PDH kinase and PDH phosphorylase are part of the mammalian PDH complex

  • Ca2+ enhances phosphatase activity (activates PDH)

  • Kinase is activated by ATP

    • High ATP → phosphorylated PDH → less acetyl CoA

    • Low ATP → dephosphorylated PDH → more acetyl CoA

Citrate synthase regulation

• Inhibited by succinyl CoA

• a-ketoglutarate is an important branch point for amino acid metabolism

• Succinyl CoA communicates flow at this branch point to the start of the cycle

Control of citrate levels via isocitrate dehydrogenase

• Inhibition of IDH leads to accumulation of isocitrate

• Aconitase is reversible, leads to accumulation of citrate

• Accumulated citrate leaves mitochondria and inhibits PFK-1 in glycolysis

Leucine

• Ketogenic

• Leucine → Acetyl CoA → Ketone bodies

• Carbons in ketogenic path will not end up in glucose

• Ketone bodies are produced by the liver and used by brain, heart, and muscle when glucose is scarce

Alanine

• Glucogenic

• Alanine → Pyruvate → Glucose

  • Pyruvate to glucose conversion is achieved via gluconeogenesis

Anaplerotic reactions

• Intermediates in TCA can be used in alternative biosynthetic pathways

• Must replenish the intermediates in order for TCA to continue

• E.g. pyruvate + HCO3- + ATP → oxaloacetate + ADP (catalyzed by pyruvate carboxylase)

• E.g. PEP + CO2 + GDP → oxaloacetate + GTP (catalyzed by PEP carboxykinase)

Fatty acid oxidation for energy (summary)

• FA Activation

  • -2 ATP (for FA of any length)

• FA oxidation: 1 FADH2 and 1 NADH per each oxidation

  • Number of oxidation cycles = (length of FA/2)-1

• Acetyl CoA oxidation: 1 FADH2, 1 GTP and 3 NADH per TCA turn

  • Number of TCA turns = length of FA/2

Activation of FA before oxidation

Fatty Acid Beta Oxidation Pathway

Beta oxidation of monounsaturated fatty acids

• Beta oxidation cycles are conducted as usual until double bond is present at C3-C4 carbon

• Delta3, Delta 2 enoyl CoA isomerase catalyzes cis to trans conversion and migration of double bond to C2-C3

  • Enoyl-CoA hydratase is stereospecific and cannot add water across cis double bond

Beta oxidation of polyunsaturated fatty acids

Requires a reduction step (with cofactor NADPH and H+) in addition to isomerization

Beta oxidation of odd numbered fatty acids

B12

• Cobalt containing vitamin

• Used in conversion of L-methylmalonyl-CoA to Succinyl-CoA in beta oxidation of odd numbered fatty acids