BMB 462Exam # 2 Study Guide Lectures: 10 - 17Lecture 10 and 11 - Begin Fatty Acid Anabolism -Tell the ‘big picture’ of fatty acid synthesis.Goal: build a 16:0 fatty acid using repeating 4 step process1. (unofficial step) Activation – of malonyl-CoA using ATP2. Condensation – activated Acetyl-CoA adds 2 carbons3. Reduction – the ketone group is reduced to a hydroxyl groupa. e- are donated by NADPH4. Dehydration – water is removed, the hydroxyl becomes an enol5. 2nd Reduction – NADPH again donates 2 e- so that the double bond is fully saturated.Metabolic and regulatory roles of acetyl-CoA carboxylase.ACC uses biotin to add carbons, since the biotin acts as a flexible swinging arm to move substrate for different (carboxylation) reactionsCitrate is the precursor for ACC functionRegulation: ACC doesn’t function w/ low glucose levels; allosteric regulation by citrate; phosphorylation/polymerization regulated by glucagon and insulin – depolymerization inhibits activityReciprocal regulation of Fatty Acid Synthesis and Degradation.Beta-oxidation is controlled by import of fatty acids into mitochondria; inhibition of the carnitine acyl-transferase inhibits beta-oxidationCitrate is a feed-forward activator of ACC, which makes malonyl-CoAMalonyl-CoA blocks carnitine acyl-transferase functionCompare and contrast sulfur containing compounds in fatty acid metabolism.a. CoA – CoA is used in fatty acid breakdown as a fatty acid carrieri. It has a reactive SH group that can form thioester bonds with fatty acid groups, which activates the fatty acids for beta-oxidation.ii. It requires pantothenic acid for synthesis (so in order to make CoA, you need pantothenic acid in your diet; it’s an essential nutrient).iii. CoA can diffuse between enzyme active sites due to an ADP handle that binds to the enzyme active site. 1. Many different enzymes bind to CoAb. Acyl Carrier Protein (ACP)i. Analogous to biotin; it tethers to the substrate and can swing it so that one reaction occurs at one active site and then it can move the substrate to another active site for another reaction.ii. Structure1. Reactive SH group; also requires pantothenic acid for synthesis. 2. Major difference from CoA – there’s a prosthetic group that covalently attached to a protein.a. In animals, this means that it is bound to the protein and then the long, stretchable “arm” can swing between active sites in big, multifunctional enzymes.i. So rather than have the substrate diffuse through the membrane and have something happen to it; it’s locked into the enzyme and can’t leave.iii. Function – to tether growing fatty acids to the enzyme1. It attaches growing fatty acids to the fatty acid synthase2. It also acts as an e- sink, drawing e- away from the active site so that chemistry can occur.c. Cysteine on Fatty Acid Synthasei. Has a reactive SH group because of the cysteine, and is tied to an enzymeii. Function: to act as a holding slot; it temporarily holds intermediates during synthesisSynthesis of Palmitate; include intermediates, enzymes, and cofactors.1. MAT (Malonyl/Acetyl-CoA-ACP transferase) adds substrates acetyl-CoA and malonyl-CoA2. KS (Beta-ketoacyl-ACP synthase) does a condensation reaction to form beta-Ketoacyl-ACP3. KR (beta-ketoacyl-ACP reductase) reduces the molecule, using NADPH as an e- donor; creates beta-hydroxyl4. Dehydration by HD (beta-Hydroxyacyl-ACP dehydratase) to generate a double bond between alpha and beta carbons5. 2nd Reduction by ER (Enoyl-ACP reductase); uses e- from NADPH to fully saturate the 4 carbon acyl6. KS returns to move the saturated fatty acid, butyryl, back to cysteine Compare and contrast fatty acid synthesis and beta-oxidation.a. Synthesisi. Location: cytoplasmii. Acyl carrier: acyl carrier proteiniii. Electron Donor: NADPHiv. D-beta-hydroxyacyl groupv. Electron Donor: NADPHvi. C2 donor: malonyl CoAb. Degradationi. Location: Mitochondriaii. Acyl Carrier: CoAiii. Electron Acceptor: FADiv. L-beta-hydroxyacyl groupv. Electron Acceptor: NAD+vi. C2 product: Acetyl CoACompare and contrast structure of fatty acid synthase.i. Vertebrates - all enzymes on 1 polypeptide1. Makes it really easy to regulate; turn on one protein and the whole thing is functioning. 2. But also reduces flexibility in production; everything is there, so the fatty acid made will only have 16:0 Fasii. Fungi – all enzymes on 2 polypeptidesiii. Plants and Bacteria - each enzyme is on a different polypeptide. 3. This gives more flexibility (part of reason plants can produce a lot more diverse FAs) 4. Also means you have to regulate everything carefully, so have different promoters to make sure you get the right amount of everythingDescribe locations of various steps in fatty acid metabolism.a. Oxidation: Mitochondria in animals, peroxisomes in plantsb. Synthesis: Occurs in the ER for both plants and animals. In plants, synthesis also occurs in the chloroplasts. In animals, it can also occur in the cytosol.c. Elongation: Occurs in the ER for both plants and animals. In animals, elongation occurs in mitochondria as well.iii. In this way animal mitochondria and ER are like bacteria (the systems probably came from bacteria)iv. Elongation is the same chemistry as synthesis process, but we have a bunch of different enzymes in membrane of ER/mito that are doing cyclic processv. Elongation is for adding Cs to 16:0 to make it longerd. Desaturation: Occurs in the ER for both plants and animals.Describe substrate, energy, and reducing power sources, and the shuttle system.NADPH from the pentose phosphate pathwayMalic Enzyme – part of shuttle system that moves acetyl-CoA out of the mitochondriaCitrate transporter moves citrate into cytosolLecture 12 Describe modification of palmitate to longer and/or unsaturated fatty acids.Elongation and desaturation typically occurs in the smooth ER.Elongation: adds 2 carbons at a time to carboxylate end of the fatty acid.Essentially uses the same 4 steps described for fatty acid synthesisDesaturation: double bonds are added 3 carbons apart, starting at position 9. In humans, double bonds can only be added to positions 4, 5, 6, and 9.Explain why some fatty acids are essential to the human diet.For fatty acids that require double bonds in positions other than 4, 5, 6, 9 i.e. linoleatePlants can add a bond to position 12, but humans can’tDescribe mixed function oxidases/oxygeneases; relate these to fatty acid desaturation.A 2nd source of e- is needed (2 e- come from
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