Biochem

• Tricarboxylic Acid Cycle


  Other names: Krebs Cycle, Citric Acid Cycle
• Reactions of the TCA cycle

  1. Oxaloacetate is first condensed with an acetyl group from acetyl CoA, and then is regenerated as the cycle is completed

  2. The entry of one acetyl CoA into one round of the TCA cycle does not lead to the net production or consumption of intermediates
• Oxidative decarboxylation of pyruvate

  1. Pyruvate, the end-product of aerobic glycolysis, must be transported into the mitochondrion before it can enter the TCA cycle

  2. Pyruvate is converted to acetyl CoA by the pyruvate dehydrogenase complex

  3. The irreversibility of the reaction precludes the formation of pyruvate from acetyl CoA, and explains why glucose cannot be formed from acetyl CoA via gluconeogenesis

  4. The pyruvate dehydrogenase complex is not part of the TCA cycle proper, but is a major source of acetyl CoA— the two-carbon substrate for the cycle
• Pyruvate Dehydrogenase Complex


  • A multimolecular aggregate of three enzymes: Pyruvate dehydrogenase (decarboxylase or E1), Dihydrolipoyl transacetylase E2 and Dihydrolipoyl dehydrogenase E3
  • The physical association of these individual enzymes links the reactions in proper sequence without the release of intermediates
• Pyruvate Dehydrogenase Complex


  • In addition to the enzymes participating in the conversion of pyruvate to acetyl CoA, the complex also contains two tightly bound regulatory enzymes, protein kinase and phosphoprotein phosphatase
• Coenzymes in the Pyruvate Dehydrogenase Complex

  E1 requires thiamine pyrophosphate, E2 requires lipoic acid and coenzyme A, and E3 requires FAD and NAD
• Deficiencies of thiamine or niacin can cause serious central nervous system problems. This is because brain cells are unable to produce sufficient ATP (via the TCA cycle) for proper function if pyruvate dehydrogenase is inactive.
• Regulation of the Pyruvate Dehydrogenase Complex

  1. The cyclic AMP-independent protein kinase phosphorylates and inhibits E1 whereas phosphoprotein phosphatase activates E1

  2. The kinase is allosterically activated by ATP, acetyl CoA, and NADH, turning off the pyruvate dehydrogenase complex

  3. Acetyl CoA and NADH also allosterically inhibit the dephosphorylated (active) form of E1

  4. Protein kinase is allosterically inactivated by NAD+ and coenzyme A, turning on pyruvate dehydrogenase

  5. Pyruvate is also a potent inhibitor of protein kinase, maximally activating E1

  6. Calcium is a strong activator of protein phosphatase, stimulating E activity, particularly important in skeletal muscle
• Pyruvate Dehydrogenase Deficiency


  • The most common biochemical cause of congenital lactic acidosis
  • Results in an inability to convert pyruvate to acetyl CoA, causing pyruvate to be shunted to lactic acid via lactate dehydrogenase
  • Causes particular problems for the brain, which relies on the TCA cycle for most of its energy, and is particularly sensitive to acidosis
• Three Forms of Pyruvate Dehydrogenase deficiency

  • The most severe form causes overwhelming lactic acidosis with neonatal death
  • A second form produces moderate lactic acidosis, but causes profound psychomotor retardation, with damage to the cerebral cortex, basal ganglia, and brain stem, leading to death in infancy
  • A third form causes episodic ataxia (an inability to coordinate voluntary muscles) that is induced by a carbohydrate-rich meal
• E1 defect


  1. linked dominant
• There is no proven treatment for pyruvate dehydrogenase complex deficiency, although a ketogenic diet (one low in carbohydrate and enriched in fats) has been shown in some cases to be of benefit. Such a diet provides an alternate fuel supply in the form of ketone bodies that can be used by most tissues including the brain, but not the liver.
• Mechanism of Arsenic Poisoning

  1. Arsenic can interfere with glycolysis at the glyceraldehyde 3-phosphate step, thereby decreasing ATP production

  2. Arsenic poisoning is primarily due to inhibition of enzymes that require lipoic acid as a cofactor, including pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and branched-chain α-keto acid dehydrogenase
• Arsenite


  The trivalent form of arsenic that forms a stable complex with the thiol (-SH) groups of lipoic acid, making that compound unavailable to serve as a coenzyme
• When arsenite binds to lipoic acid in the pyruvate dehydrogenase complex


  Pyruvate (and consequently lactate) accumulate
• Synthesis of Citrate from Acetyl CoA and OAA

  1. The condensation of acetyl CoA and oxaloacetate to form citrate is catalyzed by citrate synthase

  2. This aldol condensation has an equilibrium far in the direction of citrate synthesis

  3. Citrate synthase is allosterically activated by Ca2+ and ADP

  4. Citrate synthase is inhibited by ATP, NADH, succinyl CoA, and fatty acyl CoA derivatives

  5. The primary mode of regulation is also determined by the availability of its substrates, acetyl CoA and oxaloacetate
• Citrate, in addition to being an intermediate in the TCA cycle, provides a source of acetyl CoA for the cytosolic synthesis of fatty acids. Citrate also inhibits phosphofructokinase, the rate-setting enzyme of glycolysis and activates acetyl CoA carboxylase (the rate-limiting enzyme of fatty acid synthesis).
• Isomerization of citrate


  Citrate is isomerized to isocitrate by aconitase
• Aconitase is inhibited by fluoroacetate, a compound
• Synthesis of Citrate from Acetyl CoA and OAA

  1. Condensation of acetyl CoA and oxaloacetate catalyzed by citrate synthase

  2. Aldol condensation has an equilibrium far in the direction of citrate synthesis

  3. Citrate synthase is allosterically activated by Ca2+ and ADP

  4. Citrate synthase is inhibited by ATP, NADH, succinyl CoA, and fatty acyl CoA derivatives

  5. Availability of substrates, acetyl CoA and oxaloacetate, is the primary mode of regulation
• Isomerization of citrate


  1. Citrate is isomerized to isocitrate by aconitase

  2. Aconitase is inhibited by fluoroacetate, which is converted to fluoroacetyl CoA and forms fluorocitrate - a potent inhibitor of aconitase
• Oxidation and decarboxylation of isocitrate

  1. Isocitrate dehydrogenase catalyzes the irreversible oxidative decarboxylation of isocitrate

  2. Produces the first NADH and releases the first CO2

  3. Enzyme is allosterically activated by ADP and Ca++, and inhibited by ATP and NADH
• Oxidative decarboxylation of α-ketoglutarate


  1. Conversion of α-ketoglutarate to succinyl CoA catalyzed by α-ketoglutarate dehydrogenase complex

  2. Mechanism similar to conversion of pyruvate to acetyl CoA

  3. Releases the second CO2 and produces the second NADH

  4. Requires thiamine pyrophosphate, lipoic acid, FAD, NAD+, and coenzyme A

  5. Equilibrium is far in the direction of succinyl CoA

  6. Enzyme is inhibited by ATP, GTP, NADH, and succinyl CoA, and activated by Ca++
• Cleavage of succinyl CoA

  1. Succinate thiokinase (succinyl CoA synthetase) cleaves the high-energy thioester bond of succinyl CoA, coupled to phosphorylation of GDP to GTP

  2. GTP and ATP are interconvertible by nucleoside diphosphate kinase reaction

  3. Succinyl CoA is also produced from propionyl CoA and amino acid metabolism
• Oxidation of succinate


  1. Succinate is oxidized to fumarate by succinate dehydrogenase, producing FADH2

  2. Succinate dehydrogenase is inhibited by oxaloacetate
• Hydration of fumarate


  1. Fumarate is hydrated to malate by fumarase (fumarate hydratase)

  2. Fumarate is also produced by urea cycle, purine synthesis, and amino acid catabolism
• Oxidation of malate


  1. Malate is oxidized to oxaloacetate by malate dehydrogenase, producing the third NADH

  2. Oxaloacetate is also produced by transamination of aspartic acid
• Energy produced by the TCA cycle

  • Two carbon atoms enter as acetyl CoA and leave as CO2
  • Cycle does not involve net consumption or production of oxaloacetate or other intermediates
  • Four pairs of electrons transferred: three reducing NAD+ to NADH, one reducing FAD to FADH2
  • Oxidation of one NADH yields ~3 ATP, oxidation of FADH2 yields ~2 ATP
  • Total yield of ATP from oxidation of one acetyl CoA is 12 ATP
• Regulation of the TCA cycle

  • Regulation by activation and inhibition of enzyme activities
  • Regulation by availability of ADP
• Regulation by activation and inhibition of enzyme activities

  • In contrast to glycolysis, TCA cycle is controlled by regulation of several enzyme activities
  • Most important regulated enzymes are citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase complex
• Regulation by the availability of ADP

  1. Effects of elevated ADP: Energy consumption increases ADP, accelerating reactions that use ADP to generate ATP, most importantly oxidative phosphorylation

  2. Effects of low ADP: Lack of phosphate acceptor (ADP) or inorganic phosphate (Pi) decreases oxidative phosphorylation rate, causing NADH and FADH2 to accumulate and inhibit the TCA cycle