Cellular Metabolism II
Citric Acid Cycle
- Aerobic metabolism - Also known as Kreb’s cycle
- Amphibolic – plays a role in both catabolism and anabolism - Continuation from glycolysis
Acetyl CoA production
- Before entering the citric acid cycle, compounds are to be degraded to acetyl groups - Glycolysis occurs in cytosol
- Citric acid cycle occurs in mitochondria after the glycolysis product is synthesized
- Undergo oxidative decarboxylation by removing CO2 to produce acetyl CoA (2C) from pyruvate (3C) o Releases ONE MOLE of NADH!!!!!!!!!!
Citric Acid Cycle Reaction
- 8 reactions
- Donates acetyl CoA (C2) group to oxaloacetate (C4) Citrate (C6) - Citrate Isocitrateα-
ketoglutarate (C5)succinyl CoA
succinate
(C4)fumaratemalateOxaloac etate …
- One cycle results
o 3 NADH (2.5 ATP each) o 1 FADH2 (1.5 ATP each) o 1 ATP
o 2 mol CO2
- Electrons carried by NADH and FADH2 will be carried on the electron transport chain
o O2 reduced to H2O
Regulation in Citric Acid Cycle
- Outside the cycle
o Pyruvate dehydrogenase (Control Acetyl CoA production)
o Allosteric regulation
Inhibited by ATP, acetyl CoA and NADH
Activator: ADP/AMP, CoA and NAD+
o Covalent modification of enzyme
High ATP level, pyruvate dehydrogenase kinase is activated, causing the phosphorylation of the enzyme inactive
High glucose level, insulin activates pyruvate dehydrogenase phosphatase, the enzyme is dephosphorylated active
- Within the cycle o Citrate synthase
Inhibitor: succinyl CoA, citrate, NADH and ATP
o Isocitrate dehydrogenase
Inhibitor: ATP, NADH o α-ketoglutarate dehydrogenase
Inhibitor: ATP, succinyl CoA, NADH Cells in resting
state
Cells in active metabolic state
Need of energy little many
ATP/ADP ratio high low
NADH/NAD+ ratio high low
Central Metabolic Pathway
Catabolism
- Carbohydrates, fatty acids, amino acids enter citric acid cycle via Acetyl CoA
- Other amino acids enter through the intermediates Anabolism
- Oxaloacetate – glucose synthesis - Citrate – Fatty acid synthesis
- α-ketoglutarate and oxaloacetate – amino acid synthesis - Succinyl- CoA - porphyrin synthesis
Intermediate that has been taken out should be replaced back – Anaplerotic reaction
- Pyruvate can be converted to oxaloacetate - Catalysed by pyruvate decarboxylase - Acetyl CoA is the allosteric activator
- If there is insufficient oxaloacetate, the acetyl coA will ACTIVATE the enzyme
Glyoxylate Cycle (Modification of Citric Acid Cycle)
- In plants and bacteria (NOT animls), they can synthesize glucose from fatty acids
- Animals CANNOT convert carbohydrates to fats - Bypass the two oxidative decarboxylation of
intermediates
- This only occurs when there is excess Acetyl CoA - Routes: Isocitrate GlycoxylateMalate - Enzymes: Isocitrate lyase, Malate synthase In plants
- Occurs in glyoxysomes - Help plant to grow in dark - Seeds are rich in lipids - During germination
o Acetly CoA produced from fatty-acid oxidation - Once, photosynthesis begins, glyoxysomes disappear Yeast and Algae
- In cytoplasm
Electron Transport Chain
Electron Carriers & Electron Transport Chain
- Throughout the whole metabolism from glycolysis until citric acid cycle, the net energy yield is o 2 NADH (1 Glucose 2 pyruvate)
o 4x2 NADH +1x2 FADH2 (2 pyruvate Activation + Citric Acid Cycle) - Electron Transport chain consist of
o 4 multisubunit membrane – bound protein complexes
Complex I, II (contains succinate dehydrogenase),III and IV (Integral Membrane proteins) o 2 mobile electron carriers
Coenzyme Q (Ubiquinone)
Cytochrome c
Electrons from NADH and FADH2 transferred in series of redox reactions from one complex or carrier to the next and finally to O2. After that, the O2 is being reduced to H2O
- Protons pumped across the inner membrane to intermembrane space - Proton gradient established voltage gradient
- Can couple electron transport with oxidative phosphorylation of ADP
Electron Flow
- For NADH, a metabolic cycle will produce 10H+ (There are 10 NADH produced) - For FADH2, a metabolic cycle will produce 4 H+
- Standard free energy change for transfer of electrons o Complex I, ∆G′o =−81 kJ/mol
o Complex II, ∆G′o=−13.5 kJ/mol o Complex III, ∆G′o =−34.2 kJ/mol o Complex IV, ∆G′o =−110 kJ/mol
- Only Complex I, III and IV has the ∆G′o higher than the phosphorylation of ATP!!!!!! (∆G′o = +30.5kJ/mol) - Passing of NADH through complex I = pump 4 H+
- Through Complex III = Pump 4 H+ - Through Complex IV = Pump 2 H+ - 1
2O2+ 2H +→ H
2O (Water is produced this way)
Oxidative Phosphorylation
- Coupling process converts electrochemical potential to chemical energy of ATP o Chemiosmotic coupling
- Coupling factor (ATP synthase – F0F1-ATPase)
o Proton flows back to the matrix through proton channels in F0 unit o Flow of protons accompanied by formation of ATP in F1 unit - One proton 1 ATP
- Proton gradient exists because various proteins in electron carriers are NOT SYMMETRICALLY ORIENTED with respect to the two sides of inner mitochondrial membrane
- Proteins will take up protons from matrix when reduced and release them to intermembrane space when reoxidised
o Reactions of NADH, Coenzyme Q, O2 require protons
- Proton gradient leads to the change of conformation of ATP synthase - 3 sites
o Open (O) – Low affinity for substrate – release ATP o Loose-binding (L) – Not catalytically active, binds ADP + Pi
Tinght-binding (T) – Catalytically active, binds ATP
H+ pumps back in
ATP produced
P/O Ratio
- Number of Pi consumed in phosphorylation to number of moles of oxygen atoms consumed in oxidation - NADH – P/O=2.5 mol
- FADH2 – P/O=1.5 mol
Shuttle Mechanisms
- Transport metabolites between mitochondria and cytosol o Glycerol-phosphate shuttle
o Malate-aspartate shuttle
- Two of the NADH produced does not cross mitochondrial membrane Glycerol-phosphate Shuttle
- In skeletal muscle and brain
- For each NADH, 1.5 ATP produced in mitochondria
- Glycolysis Dihydroxyacetone phosphate Glycerol Phosphate Enter mitochondria Transfer electon to FAD to FADH2 1.5 ATP
Malate Aspartate shuttle
- In mammalian kidney, liver and heart - For each NADH, 2.5 ATP is produced
ATP Yield
- Shuttle 1: 30 ATP; Shuttle 2: 32 ATP
- When calculating ATP Yield, the following MUST be taken note: ATP Yield Calculation
- If the preparatory stage is skipped through (i.e. oxidize substances which is not in the preparatory stage of glycolysis), ADD 2 ATP!
- If ONLY ONE compound is oxidized in the payoff stage, divide the whole yield by half
- If there involve a link from Glycolysis to Citric Acid Cycle, always take the ACTIVATION OF PYRUVATE into account, i.e. 1 NADH for every pyruvate
- Apply the P/O ratio to calculate ATP yield of NADH and FADH2 - Be aware of which shuttle mechanisms is used
- If oxygen is absent, there is NO electron transport and everything involving NADH and FADH2 will be cut out!!!!!
