2 - Acetyl CoA Synthesis

• Pyruvate is at a central point in metabolism and has 5 major metabolic pathways:

  1. Transamination into alanine (amino acid synthesis)
  2. Carboxylation into oxaloacetate (gluconeogensis)
  3. Oxidative decarboxylation into acetyl CoA (energy production)
  4. Reduction into lactate (in oxygen deficiency)
  5. Fermentation to ethanol (in some micro-organisms)
• NADH (reduced form) is used in the reduction of pyruvate to lactate, releasing NAD+ (oxidised form) to be used in glycolysis to produce a small amount of ATP
• Pyruvate is oxidised to acetyl CoA and CO2
• The formation of Acetyl CoA from pyruvate is an irreversible oxidative decarboxylation reaction that occurs in the mitochondria
• The enzyme that catalyses acetyl CoA production from pyruvate is pyruvate dehydrogenase

  
• When pyruvate is decarboxylated, the 2 released electrons are transferred to NAD+ to form NADH
• Pyruvate Dehyrogenase (PDH) is the first enzyme in the citric acid cycle
• PDH is a multienzyme complex (5-10 x10^6 Da) with three separate enzyme proteins (E1, E2, E3)
• PDH is found abundant in the mitochondria of plants and animals
• The PDH-catalysed reaction required 5 cofactors (mostly vitamins) so poor-vitamin diets lead to lethargy
• PDH E1 subunit cofactor:

  • Thiamine pyrophosphate (TPP), bound to enzyme, sourced from thiamine (Vitamin B1)
• PDH E2 subunit cofactors:

  • Coenzyme A, free in solution, sourced from pantothenic acid (Vitamin B5)
  • Lipoamide (lipoic acid + lysine), bound to enzyme, not found in diet
• PDH E3 subunit cofactors:

  • FAD, bound to enzyme, sourced from riboflavin (Vitamin B2)
  • NAD+, free in solution, sourced from niacin (Vitamin B3)
• In thiamine pyrophosphate (TPP), the C* between N and S is very reactive. H+ readily dissociates to leave a C- ion that can react with the carbonyl group of pyruvate

  
• The PDH-catalysed reaction is driven by differences in electronegativity
• Lipoic acid is a very strong oxidising agent
• Lipoic acid is attached to a lysine residue in the protein to form a long extendable and movable arm made of methyl groups which allows lipoamide to react to different E subunits
• The S atoms in the disulphide bridge of lipoic acid can be reduced which causes the ring to open up (involved in the transfer of the acetyl group)

  
• In PDH there are 24 E1 subunits, 8 E2 subunits, and 12 E3 subunits

  
• Each E2 subunit is a trimer. Each monomer contains:

  1. Lipoamide arm
  2. Domain interacting with E3
  3. A transacetylase inner core (where acetyl unit is formed)
• Decarboxylation of pyruvate occurs at E1 of PDH. There is the spontaneous formation of a reactive carbon anion of TPP between N and S atoms. The carbanion adds to the carbonyl group of pyruvate to form hydroxyethyl-TPP

  
• The hydroxyethyl group of hydroxyethyl-TPP in E1 is transferred to lipoamide at E2 (transacetylation), transferring 2 electrons with it

  
• There is group transfer at E2 of PDH, transferring the acetyl group on acetyllipoamide to coenzyme A to form Acetyl CoA, leaving dihydrolipoamide

  
• Reduction of FAD and regeneration of lipoamide occurs at E3 in PDH

  
• Key reactions of PDH

  
• A multienzyme complex is significant as it:

  • Allows for co-ordinated catalysis
  • Maximises efficiency and rate of reaction by reducing side reactions due to the proximity of the enzyme subunits
  • In PDH, the flexible lipoamide arm of E2 is able to transfer the tigtly bound intermediates between sites
• Multienzyme complexes are catalytically efficient because:

  • They increase rate of reaction by increasing likeliness of collisions via minimising the distance between active sites for substrates
  • Side reactions are minimsed as complex formation allows them to "channel" metabolic intermediates between successive enzymes
  • Coordinate control ensures reactions occur in the correct order
• The conversion of pyruvate to acetyl CoA is irreversible and is a commitment to energy production
• For downregulation, high concentration of reaction products (NADH, Acetyl CoA, ATP) inhibit PDH
• For upregulation, substrates (NAD, ADP, AMP) activate the PDH complex
• Acetyl CoA production is energy demanding
• The PDH complex can be deactivated via phosphorylation of 3 serine residues on the E1 subunit
• NADH, ATP and Acetyl CoA stimulate PDH kinase activity
• NAD, ADP, AMP and coenzyme A inhibit PDH kinase activity
• PDH phosphatase activates PDH and is stimulated by increased cytosolic Ca2+ (important in muscle contraction)
• Beriberi is a disease caused by thiamine (Vitamin B1) deficiency and is a serious health problem in the Far East
• Beriberi is occasionally seen in alcoholics
• Beriberi is a neurological disorder as glucose is the primary fuel for the CNS
• Beriberi causes a build up of pyruvate and lactic acid in the blood
• Mercury and arsenite poisoning causes similar symptoms to Beriberi