Metabolic Pathways Key Concepts

• The citric acid cycle (CAC) can be thought of as a 3-stage process:

1. Oxidative decarboxylation of pyruvate, yielding Acetyl CoA
2. Acetyl CoA oxidation
3. Electron transfer and oxidative phosphorylation

Stage 1: Pyruvate → Acetyl CoA

• This stage involves 3 enzymes (E1, E2, E3) and 5 cofactors (4 from vitamins).
• The cofactors include TPP (from thiamine), FAD (from riboflavin), Coenzyme A (from pantothenate), NAD (from niacin), and Lipoate (Lipoic Acid).
• Acetyl CoA is a thioester with a high ∆G of hydrolysis.
• Two regulatory proteins: protein kinase, phosphoprotein phosphatase.
• Output includes Acetyl CoA and NADH, with ∆G'° = -33.4 kJ/mol (irreversible).
• In eukaryotes, this occurs in the mitochondria; in prokaryotes, in the cytosol.
• The Pyruvate Dehydrogenase Complex includes multiple copies of enzymes:
• E1: Pyruvate Dehydrogenase (associated with TPP)
• E2: Dihydrolipoyl transacetylase (associated with lipoate)
• E3: Dihydrolipoyl dehydrogenase (associated with FAD)
• The reactions involve oxidative decarboxylation of pyruvate to Acetyl CoA, with Pyruvate and Coenzyme A as input.
• Output: CO2, Acetyl-CoA and 2 e- in NADH
• The process works like a relay system from pyruvate to E1-TPP to E2-Lipoate to E3-FAD to NAD.
• Problems can arise from genetic mutations or thiamine deficiency (Beriberi).
• The Citric Acid Cycle takes place in the mitochondria in eukaryotes, and the cytosol in prokaryotes.
• "Balance Sheet": Input: Acetyl; Output: 2 CO2, 3 NADH, 1 FADH2, 1 ATP
• Reactions (with ∆G’° in kJ/mol, and key regulated reactions indicated):

1. Acetyl CoA + Oxaloacetate → CoA + Citrate (∆G’° = -32.2, Reg, very exergonic)
2. Citrate → Isocitrate (∆G'° = +13.3, H2O removed, then added back; endergonic) inhibits step 3 Glycolysis
3. Isocitrate + NAD(P)+ → a-Ketoglutarate + NAD(P)H + CO2 (∆G'° = -20.9 , can form NAD or NADP)
4. a-Ketoglutarate + CoA + NAD+ → Succinyl-CoA + NADH + CO2 (∆G'° = -33.5, similar to pyruvate dehydrogenase reactions)
5. Succinyl-CoA +GDP → Succinate + GTP + CoA (∆G'° = -2.9, hydrolysis of thioester strongly exergonic)
6. Succinate + FAD→ Fumarate + FADH2 (∆G' = 0, energy is captured in FADH2)
7. Fumarate + H2O → L-Malate (∆G'° = -3.8, stereospecific reaction)
8. L-Malate + NAD⁺ → Oxaloacetate + NADH (∆G'° = +29.7, energy transferred to NAD)

• The CAC releases CO2 and energy as NAD(P)H, FADH2, or GTP/ATP, and involves exergonic reactions that 'pull along' endergonic reactions.
• Energy production for Glycolysis, Pyruvate Dehydrogenation, and the CAC (NADH → 2.5 ATP's and FADH2 → 1.5 ATP's):
• Glycolysis: 2 ATP, 2 Ultimate ATPs
• Pyruvate → Acetyl CoA: 2 NADH, 3-5 Ultimate ATPs
• Citric Acid Cycle: 2 GTP, 6 NADH, 2 FADH2, 5 Ultimate ATPs
• Total ATP produced is 30-32.
• 32 ATP's @ AG'°=30.5 = 976 kJ/mol (34% of 2,840); but @ AG ~55-60, 1,850 kJ/mol (65%)
• The CAC can be either catabolic or anabolic (amphibolic pathway).
• Concentrations of intermediates are almost constant and replenished by anaplerotic reactions (Pyruvate → Oxaloacetate, PEP → Oxaloacetate, Pyruvate → Malate)
• Regulation occurs at 4 points:

1. Feeder pathway; Pyruvate → Acetyl CoA
2. Oxaloacetate → Citrate (CAC reaction #1)
3. Isocitrate → a-Ketoglutarate (CAC reaction #3)
4. a-Ketoglutarate → Succinyl CoA (CAC reaction #4)

• Regulation of exergonic reactions:
• A. by substrate availability
• B. by product accumulation/inhibition
• C. by allosteric feedback
• Allosteric Regulation:
• Lead-in to Acetyl-CoA: Inhibited by ATP and NADH, activated by CoA and NAD+, Low-E Activator Ca++
• Reaction 1: Inhibited by Citrate, Succinyl CoA, NADH, and ATP, activated by ADP
• Reaction 3: Inhibited by NADH and ATP, activated by ADP, Ca++
• Reaction 4: Inhibited by Succinyl CoA and NADH, activated by Ca++

Fatty Acids Oxidation

• Fatty Acids (FA's) are suitable as storage fuels because of high energy density, water non-solubility, and chemical inertness.
• Problems associated with FA's include the need for emulsification for transport, specialized proteins to carry them, and breaking strong bonds.
• Catabolism of FA involves:
• Digestion, mobilization, and transport from mouth or storage to mitochondria of target cells
• Beta-oxidation to Acetyl-CoA: fully saturated FA w/even number of carbons
• Oxidation of Acetyl-CoA to CO2 in the Citric Acid Cycle
• Transfer of electrons to the respiratory chain
• Digestion, mobilization, and transport include emulsification in the intestine, conversion of Triaylglycerols (TAG's) to FA's, and their incorporation into Chylomicrons.
• Mobilization and transport of stored TAG's occurs when energy is low or needed, with hormones triggering the conversion of TAG's to FA's.
• FA's are transported with serum albumins to tissues.
• Once in the target cell, FA's get to the inner matrix of the mitochondria: FA + CoA + ATP → FA-CoA + AMP + 2P, FA-CoA + Carnitine → FA-Carnitine + CoA, FA-Carnitine + CoA → FA-CoA +Carnitine
• Beta-oxidation: 4-step process involving removal of 2H+ + 2e- to FADH2 to form C=C, addition of H2O, removal of 2H+ + 2e- to NADH, and removal of Acetyl-CoA.
• The Beta-oxidation step is repeated to digest the FA.
• Products: 1 Acetyl-CoA for every 2 Carbons, 1 NADH and 1 FADH2 for every cycle.
• Each Acetyl-CoA produces 10 ATP molecules, each FADH2 ~1.5 ATP's, each NADH ~2.5 ATP's.
• Efficiency of energy extraction from FA's in vivo is around 60%.
• Digestion of mono-unsaturated FA's involves changing a cis C=C bond to a trans C=C bond, and in the production of one less molecule of FADH2.

Amino Acid Oxidation and Production of Urea

• In the liver, the amino group is removed from each Amino Acid (AA) and metabolized, and the carbon skeleton is metabolized separately.
• AA's are catabolized to digest AA's, when the diet is too rich in proteins, or when carbohydrates are not available.
• First step is the breakdown of proteins to AA's in the stomach (gastric juice, Pepsin) and small intestine (pancreas synthesizes zymogens).
• Amino acids transfer their amino group to a-ketoglutarate, forming glutamate in the blood system and transfered to the liver.
• There is a different enzyme to catalyze this reaction for each AA; all have pyridoxal phosphate (PLP) as a cofactor.
• The glutamate then undergoes oxidative deamination to form a-ketoglutarate and release its amino group (as NH4) for processing through the urea cycle.
• In muscle tissue, a-ketoglutarate and alanine are created from amino group and pyruvate
• Ways different organisms excrete nitrogen: Aquatic species excrete NH3/NH4+, terrestrial animals excrete urea, arid-climate animals excrete uric acid
• The Urea Cycle (UC) take places in the cytosol except for one reaction
• Features of the Urea Cycle include:
• Material Input: NH4+, HCO3¯, Aspartate
• Material Output: Urea, Fumarate
• Energy Exchange: 3 ATP → 2 ADP + 1 AMP + 2 P
• When fumarate returns to the CAC, one NADH molecule is formed
• The UC and the CAC are linked by a number of intermediates
• Proteins can be broken down and the carbon skeletons of the AA's used as fuels, facilitated in two levels of UC regulation:
• Long-term: the need for these fuels
• Short-term: high glutamate levels (high amino group-level)

Test your knowledge of metabolic pathways. This quiz covers key reactions, regulatory steps, and enzyme cofactors in metabolic processes such as glycolysis, pyruvate dehydrogenase complex, and citric acid cycle. Learn about metabolic disorders like phenylketonuria.