Nelson 54:TCA cycle

With oxygen around, you can get more energy than from glycolysis. Provides large reduction potential difference out of flow of electrons. Chlorophyll used for Photosystem 1 and 2. 2 produces molecular oxygen.
2: oxygen undergoes addition of electrons. Partial reduction results in reactive oxygen species (ROS). Aging may result from ROS.
ROS can damage lipids, carbohydrates, proteins, etc. Small molecule scavengers- vitamin C,E , enzymes to convert toxic compounds to less toxic. superoxide dismutase (SOD), catalase. Look in cell- where is SOD? One inside mitochondria, where respiratory activity takes place. Red blood cells have a lot of catalase to convert H2O2 to water. Oxygen is at end of electron transfer to get a 20x more ATP than from glycolysis.
3: Cycle discovered by Krebs in 1937.
4: Nature rejected paper.
6: use chemisomotic potential to generate ATP.
Note NADH and FADH2 production. Limited amount of these compounds around, required for other activities. NAD+gets depleted. Have to reoxidize NADH and FADH2. Aerobic conditions-use electron transport chain.
Key point- acetyl coA.
5: Yeast undergoes shift in metabolism from one substrate to another. coA comes from fatty acid oxidation, glycolysis pathway, amino acid breakdown. Alcohol dehydrogenase has 2 forms, one in cytosol, one in mitochondria. In humans, this enzyme is target for disulfuram. Makes acetaldehyde build up.
Compartmentation:
7:glycolysis in cytosol, TCA in matrix of mitochondria. Not watery in matrix- very few protons available. More like a colloid.
TCA is in soluble part of matrix except for succinate dehydrogenase, which is membrane bound. How do components get across outer and inner membrane?
8: Outer membrane contains porins made of beta barrels. Leaves hole in center for small molecules to enter. Hole is 10 angstroms across. Proteins up to 10,000 ml wt could enter.
Across inner membrane: tight. Can’t allow even protons to leak across.Pyruvate traansporter is one of 46 mitochondrial carriers of anionic compounds.Also found in peroxisomes. Carriers are unique to eukaryotes.
PDC- pyruvate dehydrogenase complex- has 3 subunits E1,E2, E3- names on slide 9. Cofactors:pyruvate decarboxylase uses TPP.
E2 uses lipoic acid, and coA. See slide.
10: Mammals have 60 copies E2 in complex. Bigger and spatially arranged so subunits can feed substrate from one to next. More efficient.
Regulated by phosphorylation. Has 5 copies of a kinase and phosphatase attached. Phosphorylation inactivates, dephosphorylation reactivates.
Twice as big as ribosome.
11: 1st step:
E1 removes CO2 and transfers rest to TPP.
12: Thiazolium picks up pyruvate. TPP used for decarboxylating alpha keto acids. This slide is conversion of pyruvate to ethanol in yeast. In dehydrogenase, acetaldehyde is passed to lipoic acid. CoA picks up acetyl group. Lipoic acid reduced- reoxidized by E3 enzyme. FAD picks up electrons and passes to NAD+ to make NADH.
13: Citrate is a tricarboxylic acid. CoA is released in an irreversible step.
15: Citrate synthase crystallized at different stages. Binding site for acetyl coA is created by binding oxaloacetate. ONce citronyl coA formed, hydrolysis site brought into alignment by conformational change.
14: Why go through this trouble to oxidize Acetyl coA? Cleaving to produce CO2. Nothing is wasted.
Want carbonyl on alpha carbon of citrate.
16:Aconitase moves hydroxyl stereospecifically.
Aconitase has iron-sulfur cluster in active site.
3rd step- oxidative decarboxylation.Makes alpha ketoglutarate and loses CO2. Makes NADH. 2 carbons removed are not the same ones added on from acetyl coA.
17: catalytic cycle means that if you add intermediates, reaction proceeds. Enzymes act sequentially.
4. Oxidative decarboxylation by alpha keto-glutarate dehydrogenase. E3 is same subunit as pyruvate dehydrogenase. Accomodates larger substrate.
Results in succinylcoA.
5. Succinyl coA synthetase is named as it is because it is easier to assay in reverse direction. hydrolyses off coA to produce GTP. This is substrate level phosphorylation.
6. FADH2 produced.
18: succinate dehydrogenase electrons go to ubiquinone.
19: succinate dehydrogenase upside down. Heme in bacterial form is not in mammalian form. Heme is “evolutionary relic” and not necessary. Cardiolipin- negatively charged lipid- inner mitochondrial membrane. lipid involved in making enzyme functional.
7. funarase produces malate. Fumarase found in cytosol and mitochondrial matrix. One gene with 2 locations. 2 methionines 30 AA apart. Complex import machinery- TOM complex and TIM complex- proteins that begin import get signal sequence cleaved- part goes in, part out to cytosol.
8. Malate dehydrogenase- oxidizes substrate to produce NADH. Very little oxalate in mitochondria. Mass action drives reaction. Citrate synthase pulls reaction ahead.
TCA proposed to be in complex called metabolon.Some evidence suggests channeling between active sites.Radiolabelled malate can go to citrate without influence of cold cytosolic malate, suggesting channeling. Break mitochondria open to investigate, the system falls apart. Aconitase and citrate sythase can be attached to each other.
20:how could this have evolved? Haemophilus influenza in 1995- 1st 3 enzymes were gone. The system Must be linear in this organism. Organism only grows with high concentration of glutamate to supply alpha-ketoglutarate. Lots of these products are required to synthesize other compounds. AAs, fatty acid biosynthesis, etc.
Some anaerobes have altenative oxidase.
21:Under anaerobic conditions, cycle forms 2 linear pathways with net zero energy. AcetylcoA cannot be transported directly- goes out on citrate transporter. This drains intermediates. See his notes.
22: Regulation: pyruvate dehydrogenase is highly regulated by feedback inhibition of ratio NADH/NAD+ or ATP/AMP.
Phosphorylation happens, too.
Ethanol can convert to acetyl coA. No net gain carbons. Mammals cannot convert fat to sugar. Bacteria, fungi, plants have glyoxylate cycle.
24:
no isocitrate dehydrogenase- leaves out decarboxylation.Succinate exported from glyoxisome into mitochondria. to TCA. Can be used to make sugars. Glyoxylate no decarboxylation steps. Isocitrate lyase is not in humans, but is in mycobacterium TB. and aspergillus. Identified as drug target. Would reduce therapy time.