Chapter 16 The Citric Acid Cycle: CAC Kreb’s Cycle Tricarboxylic Acid Cycle: TCA They are all the Same Cycle figured out by Sir Hans Krebs.

Key topics: To Know The Citric Acid Cycle Also called Tricarboxylic Acid Cycle (TCA) or Krebs Cycle. Three names for the same thing. Cellular respiration and intermediates for biosynthesis. Conversion of pyruvate to activated acetate Reactions of the citric acid cycle Anaplerotic reactions to regenerate the acceptor Regulation of the citric acid cycle Conversion of acetate to carbohydrate precursors in the glyoxylate cycle

Discovered CAC in Pigeon Flight Muscle Han Krebs are remarkable scientist, German by birth became English (2nd World War), later knighted by Queen Elizabeth. Lets begin first where we left off from Glycolysis with Pyruvate, which is where Hans Krebs began. (He actually worked out the urea cycle first, so this was his second, but more major scientific contribution).

Cellular Respiration Process in which cells consume O2 and produce CO2 Provides more energy (ATP) from glucose than Glycolysis Also captures energy stored in lipids and amino acids Evolutionary origin: developed about 2.5 billion years ago Used by animals, plants, and many microorganisms Occurs in three major stages: acetyl CoA production (This chapter) acetyl CoA oxidation (This chapter) electron transfer and oxidative phosphorylation (Chapter 19)

Overall Picture This needs a little editing. . . Next slide.

Overall Picture Acetyl-CoA production occurs in the mitochondria. Acetyl-CoA enters the CAC. The area blocked off all takes place in the Mitochondrion. So, first pyruvate has to get transported from the cytoplasm into the mitochondrion. In this Figure, only Glycolysis is in the Cytoplasm.

Pyr DH is a Complex Enzyme There is a lot of stuff going on here. Overall a nicely exothermic enzyme. We already know about NAD+, TPP. FAD. So lets look at lipoic acid. PyrDH is really three enzymes as a module (recurring concept).

Pyruvate Dehydrogenase Pyruvate DH a very large protein made up of three enzymes as a module. Asterix on the model is where lipoyl group is attached to E2. Model TEM

Lipoic Acid is linked to a Lys (K) You don’t have to know the structure in detail, the important point is the disulfide (oxidized state) and –SH (reduced state) and that it can form a thioester (just like acetyl-S-CoA)…next slide. What bond hooks the lipoic acid to lys?

Remember HSCoA ? from Chapter 1 Now lets put it all together, next slide. It is down here

One Unit of Pyr DH EOC Problem 6: Tests your knowledge of PyrDH. Enzyme 1 has the same name as the whole complex and is a TPP decarboxylase as well: the carbonyl (2nd carbon of pyruvate) becomes reduced from the oxidation of the carboxyl to CO2. The hydroxyethyl-TPP then reduces lipoate and is transferred to CoA-SH to form acetyl-S-CoA and completely reduced lipoate. Reduced lipoate then through FAD-enzyme 3 reduces NAD+ to NADH. Ahhhh, done. EOC Problem 6: Tests your knowledge of PyrDH. EOC Problem 7: Thiamin deficiency and blood pyruvate.

Pyr DH is a Cool Enzyme The overall is what you need to know. Pyr-DH is in the mitochondria…and now lets get the Acetyl part of pyruvate into the CAC. EOC Problem 5: NAD+ in oxidation and reduction reactions (a through f should be easy).

Here is the Whole Cycle. All cyclic pathways have to have an acceptor molecule, here oxaloacetic acid (OAA) for the input, here acetyl part that come from pyruvate. We will do each enzyme separately, but look how it is put together shading the input acetyl carbons so that during this “turn” of the cycle, the CO2 that comes off (circled here in red) do not come from the acetyl carbons, but from OAA (oxaloacetic acid) the acceptor. The other outputs from the cycle (besides CO2) are energy metabolites: 3 NADHs, 1 FADH2 and a GTP. The GTP is the same worth as an ATP. So as the cycle spins it has all these outputs. CO2 is a metabolic end product and a waste from heterotrophs, but a carbon source of photosynthetic and other autotrophs. The NADHs and FADH2 will be oxidized (in the mitochondria) by the electron transport system, producing the proton motive force to produce ATP (we will do this later in Chapter 19). Now lets look at each CAC enzyme.

Citrate Synthase Convention to write incoming Acetyl on Top Citrate synthase attaches the acetyl group onto OAA to form CITRATE which is a tri-carboxylic acid (3 pKa’s !!) and a molecule that looks pretty symmetric but as we will see later, it is not, but pro-chiral. Note that water is important: the oxygen of water ends up as the second oxygen of the acetyl carboxyl group. The one of the hydrogens of water is on the sulfhydryl of CoA-SH. This is a nicely exothermic reaction. EOC Problem 32, further on the thermodynamics of Citrate Synthase.

Aconitase, the Ferris Wheel Calling this a ferris wheel is a (lame?) biochemical joke. The enzyme removes a water and then adds another back to effectively transfer the citrate OH to the lower 2 carbon unit making isocitrate. The reaction is slightly endothermic, but right next to an exothermic push, citrate synthase. To do this the enzyme utilizes an iron-sulfur complex (hence the ferris wheel idea), see next slide.

The Aconitase Iron Sulfur Complex The iron sulfur complex (we will see some of these in the respiratory electron transfer system) is held in place by cysteines. Aconitate is the intermediate after a water is removed, and for some reason is included in the Overall Cycle and really doesn’t have to be.

Aconitase has More than One Role Mitochondrial aconitase: Citric Acid Cycle Cytosolic aconitase: 2 roles: 1. citrate  isocitrate 2. iron response regulator Effect of IRP1 and IRP2 on the mRNAs for ferritin and the transferrin receptor. Aconitase has its role in CAC, but it also “moonlights” in the cytoplasm as a source of NADPH (this will make sense after we see what isocitrate DH does) and as a regulatory protein regulating iron metabolism. With high iron in the diet, ferritin protein needs to be made to store iron (mainly in the liver) as ferritin, with low iron the liver needs to make transferrin to bind low amounts of iron (tranferrin a serum protein has one of the smallest Kd s known…so it has a tremendously high affinity for iron which it delivers to all tissues).

Aconitase binding iron/RNA To become an iron response regulator, aconitase changes it shape (due to lack of iron) so it can bind RNA. BOX 16-1 FIGURE 2 Two forms of cytosolic aconitase/IRP1 with two distinct functions. (a) In aconitase, the two major lobes are closed and the Fe-S cluster is buried; the protein has been made transparent here to show the Fe-S cluster (PDB ID 2B3Y). (b) In IRP1, the lobes open up, exposing a binding site for the mRNA hairpin of the substrate (PDB ID 2IPY)

Isocitrate DH ΔGo’ = -21 kJ/mole Mn++ cofactor Isocitrate DH is also a decarboxylase and has manganese as a cofactor. You only need to know the overall reaction which is producing: CO2, NADH, and α-keto-glutarate (a 5 carbon dicarboxylic acid), and it is nicely exothermic. Mn++ cofactor EOC Problem 8 is all about IsocitDH.

αKG DH is Just Like Pyr DH TPP, lipoate FAD We don’t have to go through the enzyme-1, enzyme-2, enzyme-3…but it is just the same as Pyruvate DH having a lipoic acid attached to a lysine, TPP, FAD. Again another nicely exothermic enzyme producing the second NADH, a CO2 and succinyl-S-CoA.

Succinyl CoA Synthetase : Substrate Level Phosphorylation Look at the name of this enzyme: it is named for the reverse reaction and it is not a synthase but a synthetase (an ET has sneaked in). The sythetases always involve an nucleotide triphosphate, the synthases don’t (such as citrate synthase). This is an equilibrium reaction, very small ΔGo’…but it is a substrate level phosphorylation (like 3-P-glcyeraldehyde DH in Glycolysis). The end product succinate is perfectly symmetrical: this is a very important point when we look at which carbons come off as CO2 in experiments. One GTP = One ATP Nucleoside diphosphate kinase: GTP + ADP  GDP + ATP ΔGo’ = 0

Succinate DH = Old Yellow SuccDH is called “old yellow” because it was one of the first FAD enzymes isolated and studied quite well in the middle of last century. The enzyme is well connected to the electron transport system (Chapter 19), and an equilibrium reaction almost perfectly. See that fumarate is trans.

Malonate was One of the First Competitive Inhibitors Known So you know for sure what a Lineweaver-Burke plot of this enzyme with and without the inhibitor looks like.

Fumarase: the addition of water in two parts Fumarase is also a near equilibrium enzyme and forms Malate (alpha hydroxy 4 carbon dicarboxylic acid)…and next the LAST reaction of the cycle, but first we need to see the difference between malate and maleate, next slide.

Don’t Confuse Malate and Maleate Maleate is used as a buffer because it is not a metabolic intermediate, but the cis form of fumatare.

Malate DH is Endothermic WOW, this is like aldolase in Glycolysis, the ΔGo’ is largely endothermic and must be overcome so that the cycle will work, next slide

CAC Energetics See how the front part of the cycle pulls and pushes the cycle along, reactions 5-8 are equilibrium reactions regardless of their ΔGo’. Now lets watch where the carbons go, next slide.

Watch Where the Label Goes Now we will see that citrate is pro-chiral. EOC Problem 18: Labeled glucose carbons and where they go in CAC.

Citrate is Prochiral This means that citrate can only fit into aconitase in one specific orientation, so that the acetyl carbons from pyruvate do not come off as CO2 during the first turn of CAC.

The Acetyl Portion does not get oxidized to CO2 Until the Second Round Thus, if we put in 100 molecules of raditoactive acetyl-S-CoA, none of the radioactivity will come off during the first round. And, during the first round half the ratioactivity will be at either end of succinate, so on the second round only 50% of the label will come off as CO2, then 25% at the 3rd round, 12.5% on the 4th…and so on. And it gets randomized at Succinate

Energetics of Glycolysis and CAC in ATPs Starting with glucose and completely oxidizing it to CO2 produces a lot of ATP (biochemistry money). We will see why later, but for now an NADH is worth 2.5 ATP and FADH2 is worth 1.5 ATP. This only has the energetic enzyme steps: one molecule of glucose produces 30-32 ATPs. Compare this to fermentations: these need to recycle the NADHs, so they only get 2 ATPs from glucose  fermentation end products (we did lactic acid, and the yeast CO2 and ethanol pathways). Other fermentations produce only 1 ATP per glucose. It is obvious that the complete oxidation harvests more ATP than fermentation, but fermentation and complete oxidation work at similar efficiencies: the fermentation end products have energy that has been untapped. Further fermentation end products have other biological effects and for many organisms in anaerobic environments is the only way to generate ATP (substrate level phosphorylation). Now, we will see why a “modified” CAC has to be in anaerobic organisms. Next slide. EOC Problems 1 and 2: Balanced equations for Glycolysis and CAC.

CAC in Anaerobic Not-Respiratory Organisms It’s a 2 input FORK This is because all organisms need to make α-ketoglutarate which is the starting metabolite to make the amino acids glutamate, glutamine, proline and arginine, and that succinyl-S-CoA is the starting metabolite to make porphyrins (hemes, cytochromes, chlorophyl). The slide also includes nucleotides because glutamine is a prime nitrogen donor in nucleotide synthesis. These organisms lack the enzyme α-ketoglutarate DH, so these enzymes can not work as a cycle but rather only for biosynthesis. Also OAA is the starting metabolite of aspartate, asparagine, threonine, lysine, isoleucine, methionine.

This is Why This is the biosynthetic output of CAC and reactions just prior to CAC. OAA  D, N, I, K, T, M

Anaplerotic Reactions Anaplerotic reactions are “filling up” reactions. So, as biosynthesis pulls off CAC intermediate, they need to be replaced. The replacement reactions form OAA or malate from pyruvate or PEP. A cyclic pathway can not work without replenishing the acceptor molecule, OAA.

Regulation of CAC EOC Problem 30 and 31 on oxygen and NAD regulation of CAC. The regulation of CAC (all allosteric) is at the drivers of the cycle (exothermic reactions). Both positive (green) and negative (red) regulation: molecules that signal low energy level signal the cycle to go faster; molecules that signal energy levels high signal the cycle to slow down.

Pathway Proteins Form Functional Units but It’s Concentration Dependent Dilution of a solution containing a noncovalent protein complex—such as one consisting of three enzymes—favors dissociation of the complex into its constituents. This is just to emphasize that in the cell, these enzymes are made in large enough concentrations so that they function as modules.

Pathways are Protein Modules Flagella LPS Outer Membrane Peptidoglycan Cytoplasmic Membrane Glycolysis ATPase RNA

In Animals CAC can not be used for Gluconeogensis from Ac-SCoA D, N, L, K, M, T, I Gluconeogenesis (Chapter 14) can not begin with acetyl-S-CoA: converting any CAC intermediate to PEP would deplete the CAC of the receptor molecule and stop glycolysis. Some organisms can do this, but for that they need a way to produce CAC intermediate from acetyl-S-CoA :: it is called the glyoxylate cycle or glyoxylate shunt. Porphrins: heme (cytochromes, hemoglobin), chlorophyll E, Q, P, R

In Bacteria and Plants, Not Vertebrates Overall: 2 Ac-SCoA  Succinate Succinate  OAA These are the only ones that can use acetyl-S-CoA to make glucose (thus making both gluconeogensis ana PPP working biosynthetically from acetyl-S-CoA). The importance of this is that the degradation of many nutrients, particularly fatty acids, results in producing acetyl-S-CoA. The cycle only has two new enzymes: isocitrate lyase and malate synthase. Isocitrate lyase essentially breaks the bond that connects the top part of isocitrate (which looks like succinate) to succinate and glyoxylate (the proton of the alcohol goes to succinate) oxidizing that carbon to a carbonyl. Points to remember is that this cycle takes 2 acetyl-S-CoA inputs and produces 1 succinate that can regenerate the CAC acceptor molecule OAA. This allows OAA in these organisms to supply gluconeogenesis BECAUSE it can be replaced by the Glyoxylate Cycle. NADH and FADH2 Oxaloacetate CAC

Glyoxylate Cycle in Plants in a Membrane Body In plants the glyoxylate cycle has its own organelle.

Linkage to Gluconeogenesis in Plants This is important because many plants store energy as oils (triacylglycerols; where do all the kitchen cooking oils come from?).

Regulation Linkage You don’t have to know the linkages and their regulation, but you do have to know that these like all metabolic pathways are highly regulated.

Things to Know and Do Before Class Pyruvate DH…all three parts and cofactors. Chemistry of each step in Citric Acid Cycle. Overall CAC thermodynamics (which steps are at Eq and which are drivers. Prochiral nature of citrate. Amphibolic nature of CAC and why fermenters need almost all of CAC. Importance of anaplerotic reactions and how they work. Glyoxylate Cycle (mammals lack) but plants, some invertebrates and bacteria have it. What does it do? EOC Problems 1-9, 16, 18, 19, 30-32.