BICH 411 1st EditionExam # 2 Study Guide Lectures: 6-11Lecture 6 (September 23)-The TCA (also called Citric Acid Cycle and Krebs cycle) is the next step in metabolism. It produces ATP (from GTP), CO2, and cofactors NADH and FADH2. It consists of 8 steps plus a “step0” that converts pyruvate (made in glycolysis) to acetyl-CoA. This occurs in the mitochondria. The NADH and FADH2 cofactors carry the electrons to the electron transport chain. **The TCA cycle is amphibolic (both catabolic and anabolic).Reaction 0: pyruvate + NAD+ + CoASH acetyl CoA + NADH + H+ + CO2 with the help of a pyruvate dehydrogenase complex (made of three enzymes: E1, E2, and E3. This is similar to the alpha-ketoglutarate dehydrogenase mechanism.) In the process, a CO2 is released and a NADH isformed. ***Be sure to know the functions of the enzymes. E1 transfers the hydroxyethyl groupfrom pyruvate to TPP, forming HETPP (hydroxyethyl TPP). E2 transfers the hydroxyethyl group to lipoic acid where it is then oxidized to form acetyl dihydrolipoate. E3 facilitates the transfer of the hydroxyethyl group to CoA. NAD+ picks up a hydrogen, reoxidizing lipoic acid, and reducing NAD+ to NADH + H+. **E3 restores E2 through it’s reoxidation of lipoic acid. **5 coenzymes are required: TPP, Riboflavin (FAD – transfers electrons), Niacin (NAD+ transfers hydride in the complex), Pantothenate (in CoA), and Lipoic acid (which carries electrons and bridges the complex with it’s long arms). **It’s important to recognize that this reaction is irreversible!**Also, note that Acetyl-CoA, ATP and NADH are allosteric inhibiters here. NAD+ and CoA are positive allosteric regulators.Reaction 1: Oxaloacetate + Acetyl-CoA  Citrate, with the help of citrate synthase. **This is a claisen ester condensation** NADH, ATP, and succinyl CoA are all allosteric inhibiters in this step. This reaction makes a C-C bond.Reaction 2: Citrate  Isocitrate, with aconitase. This is an isomerization reaction. **It removes the pro-R H from the pro-R arm of citrate. *there is an iron-sulfur cluster in aconitase. **It’s important to note that fluoroacetate blocks the TCA cycle. It blocks the cycle by inhibiting aconitase, preventing the isomerization of citrate. It’s very poisonous!Lecture 7 (September 25)Reaction 3: Isocitrate + NAD+ alpha-ketoglutarate + NADH + CO2, with isocitrate dehydrogenase as the enzyme. This is a redox reaction, collecting electrons for the electron transport chain. It’s important to note that the CO2 carbon comes from Carbon 2 on isocitrate.ATP and NADH are negative allosteric regulators here (inhibitors) while ADP is a positive allosteric regulator (activator).Reaction 4: alpha-ketoglutarate + CoASH + NAD+  succinyl-CoA + NADH + CO2, using alpha-ketoglutarate dehydrogenase as the enzyme. This is a redox reaction. This mechanism is very similar to the pyruvate dehydrogenase complex, but E1 and E2 are different. This also uses five coenzymes. AMP is a positive allosteric regulator, while NADH and succinyl-CoA are negative allosteric regulators.Reaction 5: succinyl-CoA + GDP + Pi + H2O  Succinate + CoASH + GTP, with succinyl CoA synthetase as the enzyme. This is substrate-level phosphorylation. **The GTP is easily convert to ATP (the only ATP produced per cycle) with the enzyme Nucleoside Diphosphate Kinase. **Note how Histidine is involved in this reaction**Reaction 6: Succinate + FAD  fumarate + FADH2, using succinate dehydrogenase. This is a redox reaction. **FADH2 carries 2 electrons, both from carbons. **This is the only part of the TCA cycle that is also part of the electron transport chain (these electrons go to coenzyme Q (UQ). Reaction 7: Fumarate + H2O  L-Malate, using Fumarase. **Trans addition of the water molecule** This is an isomerization reaction. There are 2 possible mechanisms for this reaction. One mechanism has a carbonium ion with a positive charge, and the other has a carbanion witha negative charge. Reaction 8: L-malate + NAD  oxaloacetate + NADH with malate dehydrogenase. **note CO2 isn’t produced here. This is a redox reaction. This is a very unfavorable reaction but citrate synthase is able to pull this reaction forward and restart the cycle. **It’s important to note that the NAD+-dependent enzymes are very stereospecific (pro-R or pro-S arm). To summarize the TCA cycle, 2 CO2 molecules, one ATP, and four reduced coenzymes are produced for each cycle. **review the equations on page 625-626 for the stoichiometry to make sure you understand the “why’s” – this could be a good test question!-Anapleurotic reactions - produce intermediates for the cycle. Ex: Pyruvate carboxylase (**mostimportant one**), Malic enzyme. **PEP carboxylase isn’t really an anaplerotic reaction, so if question on test says “all but one are anaplerotic” this is that one!** Anaplerosis is important in diabetes. These reactions are causing the release of insulin. Exercise can increase this and restore insulin activity (this is why exercise can occasionally reverse diabetes).-reductive TCA cycle is reverse cycle, occurs in some bacteria and archaea. (CO2 is used instead of produced)There are 3 main sites of regulation in the TCA cycle: the citrate synthase step, the isocitrate dehydrogenase step, and the alpha-ketoglutarate dehydrogenase step. They are energetically favorable. ***Phosphorylation (kinase) inactivates the pyruvate dehydrogenase reaction. Dephosphorylation (phosphatase) activates this reaction.-Glyoxylate cycle is beneficial for algae and bacteria. This cycle is also the reason seeds can growin the ground without sunlight. Review this concept. Lecture 8 (September 30)FADH2 and NADH carry electrons through protein complexes. Eventually, this will create a protein gradient in the mitochondria. There are 4 protein complexes in the inner mitochondria membrane. -Ubiquinone (UQ) helps carry electrons and is hydrophobic, so it stays in the core of the membrane bilayer. Cytochrome C also carries electrons. **Electrons always go toward more positive reduction potentials****it’s important to note that complexes I and II have lots of Fe/S centers!!**For table 20.1, know which prosthetic groups (coenzymes) go in which complex and how theywork***Figure 20.3 is important to know for the exam. The electrons in complexes I and II are collected through catabolism. Complex IV doesn’t have any Fe/S centers!! (good test question) Protons ultimately moved to O2 to form H2O. (2