Review with Nelson

Look at space he alotted in notes for emphasis on exam. He won’t use the particular questions in the practice exam again. 6 lectures were 1.5 hrs each.
Starting with TCA Cycle: looking at big picture, know pyruvatedehydrogenase in detail and how subunits work. What is process with dihydrolipoylgroup? Look at similarities pyruvate dehydrogenase and other dehydrogenases and glycine cleavage system. TCA cycle is closed circle generating NaDHand FADH2, passed on to ETC to follow through redox centers,- to+ potential- energy captured in proton gradient.
Real insight was Peter Mitchel and chemiosmotic hypothesis. Use proton gradient, not high-energy intermediate.
Distinguish substrate level and oxidative phosphorylation. Mitchel proposed that inner mitochondrial membrane was imperm to protons to build up gradient.
TCA and other pathways- 1st 3 steps similar to other pathways. When pathways of AA mimic TCA, be aware.
TCA is generating NADH as produced. Cycle cannot cont w/o reoxidizing NADH- run out of substrate. Passed along to ETC complex1-> flavin NADH is obligatory 2electron donor, cannot reduce iron center. Flavin can take 2 and pass on one electron at a time.
Think about TCA depending on reoxidation NADH, depending on ETC.Some bacteria have other ways- some organisms miss parts of TCA, like Haemophilus. Influenza runs pieces of the cycle linearly in 2 directions. malate dehydrogenase to fumarase ran backwards to make succinyl coA to make heme. Consumes NADH from other side.
TCA is source of building blocks for other biosynthetic processes, alpha-ketoglutarate, oxaloacetate, etc. Asp in nucleic acid biosynthesis and urea cycle. Be familiar with anaplerotic reactions like pyruvate carboxylase to regenerate oxaloacetate.
Glyoxylate cycle- protists and plants and bacteria bypass decarboxylating steps in cycle. We cannot convert 2-C substrates into sugars because there is no net gain of C in TCA. Organisms that make sugars from Acetyl CoA do so by glyoxylate shunt. Isocitrate lyase yields glyoxylate and succinate. Glyoxylate +Acetyl CoA yield malate to continue the cycle. Target for drugs.
ETC lecture
FADH2 produced in succinate dehydrogenase (complex2)enzyme with dual role. FADH2 used instead of NADH because electrons do not have thermodynamic force to make NADH. Bypass complex 1- miss 4 protons- less ATP if you start with complex 2.
I large membrane sector with 7 subunits, arm in matrix has redox centers, flavin, iron sulfur centers, and ubiquinone. Keep movement of electrons through complexes in mind.
uQ binds to get reduced and oxidized in Q cycle.
bc1: Rieske FeS center and BL heme split path of electrons. one to cytochrome c1 and cytochrome c, one from Bl to BH, to UQ. Cycle repeats to reduce second ubiquinone at N center. Protons on uQ pumped out across membrane, driven by ability of structure to capture energy when electrons move.
complx4- electrons carried on cytochrome c. Found every organism, almost. Structure conserved. Heme in it to carry 1 electrons. Binds cytochrome C structure- to cytochrome c oxidase. Be fam w structures in 4, copper a and b binuuclear center. Oxygen binds betw Feand Cu. Protons to make water are chemical protns. Pumped ones are distinct. Certain number common inhibitors used- rotenone I, antamycin A , III, cyanide IV, be aware of where list of them in notes works.
ATP synthase: complex molecule. Be able to sketch bacterial structure with FO and F1, ball with alpha, beta, delta. b not crystallized.Through center- axle is gamma. Associated w epsilon. Stalk through ball toward membrane surface. C subunits form ring with 2 transmembrane segments each, with asp carboxyl important to the mechanism. Protons protonate the carboxyl group. know elevator model. Force of proton gradient turns 1/10 or 1/12 rotation. As proton goes around, finds exit channel to matrix. Be familiar with binding change mechanism. 3 conformations active site on beta subunit: Loose, tight, open. Conformations are interchangeable. Turning gamma distorts alpha and beta to change conformation. Change in conformation is responsible for releasing the ATP tightly bound at the tight site. Phosph bonds formed spontaneously and becomes tightly bound- force of H gradient needed to release ATP.
Remember reconstituted vesicle experiment with bacteriorhodopsin. Synthesize ATP with light to cause proton gradient in vesicle.Proved no high energy intermediate.
Evidence to prove ATP synthase turned- used Cys engineered in molecule between gamma and beta- oxidized-disulfide formed-could not turn. Reduce- restore function (wont ask about this). More elegant- beta with histidine tag on N terminal to anchor ATP synthase to nickel coating, held subunit down. Attached actin filament to gamma using biotin and avidin. Could see turning.
P/O ratio- consists of the number of ATP made for every electron. Look complex by complex. Complex 2- no protons. 14 p+per pair electrons. 1:4,3: 4,4: 2. ATP synthase produces 3 ATP per turn. 12 subunits- 12 protons required. 4 protons per ATP. 10= 3.3 protons per ATP. not much data.
Be aware of mitochondrial carrier proteins in membrane to translocate small negative molecules ATP, phosphate, FAs, malate, asp, alpha-ketoglutarate, etc. ATP must come out.Exchange ATP/ADP is high rate.
Similar amount phosphate through.
Metabolism AAs- 40 pathways. Do not memorize or draw structure.
Look at emphasis in notes- similar pathways to TCA, source of N from transamination, PLP involved in exchange, have glu dehydrogenase to remove amine from backbone- dehydrogenase removes and leaves as ammonia.
Basic principle- most compounds come from few starting compounds from glycolysis and TCA- break down to compounds in TCA. Keep intermediates in mind. Synthesis- break down into families based on starting elements- 5 families. See lecture for details.
Common features- activate carboxyl group by adding phosphate, AMP, succinyl coA.
Synthesis proline is different- cycle formed in an aldehyde reaction with amine at alpha carbon. alpha-ketoglutarate side chain carboxyl activated, Schiff base, forms ring. Synthesize arg, same method- acetylate alpha-amino group to protect- make ornithine, take acetyl group off.
Feature- metabolism of small molecule- easier to build uup, then break down. Gly up to Ser, then break down Ser. Where else does this happen? Carboxylate and split down middle.
Aromatic AAs- we do not have shikimate pathway. Be aware of herbicide Roundup and what it inhibits.
Metabolons predicted to exist, hard to isolate. Multiple enzymes exist in pathways, assembled into complex to channel substrate through. No release into media, evidence for channeling. Seen in trp pathway. Some genes fused, proteins coupled with disulfide bonds.
Reguation of pathways- complex branching- where does regulation take place? at branchpoints. end products feed back to inhibit at branch points. alternate ways to regulate in different organisms- aspartyl kinase in bacillus subtilis vs insects. chart of how differences in regulation look is in notes.
Urea cycle- 2 compartments- 2 steps in mitochondrion, 2 in cytosol in mammals. Dont worry about yeast. Synthesize carbamoyl phosphate in mitochondrial. transporters move ornithine in and citrulline out.Interesting step- Consumes ammonia- one N comes from Asp. Remember names of compounds. Split off fumarate- seen in purine and pyrimidine metabolism. Urea eliminates N in some mammals. Concentrated form of nitrogen. Live in air, cant diffuse out ammonia. For animals with weight concerns, take to uric acid to excrete as solid and not waste water (4Ns). CPS is distinct in cytosol (pyrimidine metabolism- uses gln for N) and mitochondrion (urea cycle).
Breakdown AAs- often see dioxygenases. 2 oxygen into product. Monoxygenase look like cytochrome p450- one oxygen in molecule, one to water. Used to break rings in degradation pathways.
Human diseases- have in mind as material for questions.Which enzymes causes which disease.
Branched chain- no questions.
Nucleic acid metabolism
De novo synthesis pathways in some detail- talk in meaningful way in terms of where inhibition act, defects in regulation, PRPS1 role in feedback regulation- can be overactive- too much purine- gout.
Causes of gout- PRPP activates pathway, oversynthesize purines with too much urate, precipitates in joints, causes gout. Pathway features- where Glu used, 2 sites of N10 formyl tethydrofolate as donor, affected by methatrexate. Talk about where impacts are on synthesis. Know GPAT, which affected by Gln and hydrofolate. Branch at IMP to AMP and GMP. See regulation of this. To make GMP requires ATP, ATP requires GTP. Allows uniform making of molecules.
Adenylosuccinate lyase- important to formation AMP, involved in splitting off fumarate in another rxn. as well. Disease associated is an autism disorder. Breakdown purines- costly to make, so salvage pathways used. HGPRT used. couples hypoxanthine and guanine w PRPP. Absence produces Lesch Nyhan syndrome.
Breakdown- adenosine deaminase in SCID. If compound builds, inhibits ribonucleic acid reductase which makes DNA nucleic acids. Block purine pathway, intersecting pathways affect each other.
Be aware of allopurinol. Analog hypoxanthine.Converted to inhibit xanthine oxidase, Treat gout.
Pyrimidine synthesis pathway- Gln nitrogen source. UMP has to be converted to TMP to make dTMP for DNA. Long and indirect path. Enzymes is thymidylate synthase. Look it up.Uses N5N10 tetrahydrofolate, converted to dihydrofolate, regenerate tetrahydrofolate by dihydrofolate reduction, which is inhibited by methotrexate. Treat infection and cancers by inhibiting DNA synthesis
Be aware of how bifluorouracil works.
see notes.
Azoserine and DON, too. AZT is analog of thymidine- interferes with reverse transcriptase. Incorporate- no 3’OH.Resistance develops to this compound, so there are inhibitors for proteases to process protein to make virus and other reverse transcriptase inhibitors. Look at notes. Will not pick obscure facts.