Metabolism II PDF 2022

5 Dental student Asma’a Abu-Qtaish Nafez Abutarboush (Krebs, Citric acid, TCA) cycle In the last sheet, we began discussing Krebs cycle, we focused on the electron carrier it produces (NADH & FADH2). Today, we'll get into this pathway in more detail. Before we start, let's highlight the three names...

5 Dental student Asma’a Abu-Qtaish Nafez Abutarboush (Krebs, Citric acid, TCA) cycle In the last sheet, we began discussing Krebs cycle, we focused on the electron carrier it produces (NADH & FADH2). Today, we'll get into this pathway in more detail. Before we start, let's highlight the three names of this cycle and the reason behind each: 1. Krebs Cycle: Named after Sir Hans Krebs, the scientist who discovered it. 2. Citric Acid Cycle: Named for the first intermediate produced in the pathway. 3. TCA Cycle (Tricarboxylic Acid Cycle): Citric acid, the initial product, contains three carboxylic acid groups, making it a weak acid but stronger than others due to its three protonated or dissociable groups. - Krebs Cycle includes 8 steps, 8 reactions, 8 enzymes, and 8 products (we have to memorize all of them). - Naming a reaction after a molecule can occur for several reasons: 1. It is the starting molecule 2. It is the final molecule 3. It is an intermediate molecule with important function - Citric acid ( ‫ )ملح الليمون‬is used in Soft drinks and Cleaning (since it can bind with Ca+2) The second 4 reactions The first 4 reactions - The final product in breaking down proteins, lipids and carbs is Acetyl CoA which starts this cycle. - We will divide the cycle into two halves. The first four reactions are involved in removing the two carbons that enter the cycle from acetate. The second four reactions are dedicated to reforming the four carbons to regenerate the molecule it starts with (oxaloacetate). - This sentence can help you remember the molecules involved in this pathway: "CIA Sent Soldiers For My Office." → Now, let's get deeply into each step of the Krebs cycle: 1. The cycle begins by combining acetate (2 carbons) with oxaloacetate (4 carbons) to form citrate (6 carbons). This reaction requires energy, which is obtained from the breakdown of Acetyl-CoA into acetate and CoA - This reaction is catalyzed by an enzyme called citrate synthase. 2. Citrate (6 carbons) converts to its isomer and this conversion is catalyzed by an enzyme called aconitase. This conversion is necessary because to extract energy from food, we must break down molecules into smaller units through oxidation. Citrate contains 3 carboxylic groups (which cannot be oxidized) and a hydroxyl group (tertiary alcohol that also cannot be oxidized). Therefore, to enable oxidation, we convert citrate to its isomer. 3. Isocitrate (6 carbons) is a secondary alcohol, so it can be oxidized. In this step, we first oxidize isocitrate, then release one carboxyl group from it in the form of CO2. Since oxidation and decarboxylation occur here, we call this reaction an oxidative decarboxylation reaction. NADH is also produced to carry the electrons, making the first dehydrogenation in the pathway. - This reaction is catalyzed by isocitrate dehydrogenase. One of the H will come out as H+ and the other H- which bind to NAD+ to produce NADH. 4. Alpha-ketoglutarate (5 carbons) is produced. Here, our aim is to release the other added carbon. We convert alpha-ketoglutarate into succinyl-CoA by removing the terminal carboxyl group and transferring the electrons to NAD+. After removing the terminal carboxyl group, the carbonyl group becomes the new terminal, which is unstable, so CoA binds to it to give stability. - This reaction is catalyzed by alpha-ketoglutarate dehydrogenase. 5. Succinyl-CoA brings us back to the initial number of carbons (4 carbons), but we need to make it similar to the starting molecule (Oxaloacitate). First, we remove CoA, and the energy produced in this process is used to bind Pi to GDP, producing GTP, which is the only energy molecule produced in this pathway. This reaction is catalyzed by succinate thiokinase. Now, we examine succinate (4 carbons) and find that its two terminals are the same as oxaloacetate. Thus, we just need to reform the carbon in the middle, we have to convert CH2–CH2 in succinate to keto group. To understand the process, we will explain it reversely by converting the keto group in oxaloacetate to CH2–CH2 (Alkane) in succinate: ▪ We convert the keto group into an alcohol group by reduction. ▪ We convert the alcohol group into an alkene by dehydration. ▪ We convert the alkene into an alkane by reduction. 7 6 6. So firstly we convert succinate into fumarate (CH2-CH2 to alkene) by dehydrogenation (oxidation), here we will use FAD as the electron carrier to produce FADH2. - This reaction is catalyzed by succinate dehydrogenase. 7. We convert fumarate into malate (alkene to alcohol) by adding water to the double bond in the alkene (hydration), this reaction is catalyzed by fumarase. 8. The final step in reforming our first molecule is converting malate into oxaloacetate (alcohol to keto group). This is done by oxidation, and here, the final dehydrogenation takes place, this reaction is catalyzed by malate dehydrogenase. The net product of the reaction: 3 NADH +1 FADH2 + 2 CO2 + 1 GTP (or ATP since they have almost the same free energy value) The enzymes: 1. Citrate synthase (from its name, it synthesizes the citrate) 2. Aconitase “aconitate”) (due to the intermediate 3. Isocitrate dehydrogenase 4. α-ketoglutarate - Keto→ keto group, -gluta → 5 carbon, and -ate → an acid nature 5. Succinate thio-kinase - thio→involvement a thiol group, kinase → addition of Phosphate group 6. Succinate 7. Fumarase 8. Malate dehydrogenase → How many dehydrogenations are in the reaction? 1. In the third reaction (6 carbons to 5 carbons). 2. In the fourth reaction (5 carbon to 4 carbon). 3. In the sixth reaction (Alkan to alkene). 4. In the eighth reaction (alcohol to ketone group). Every Dehydrogenase in their mechanism gain electrons and put them in NAD+ except of Succinate dehydrogenase put them in FAD - All the molecules in this cycle randomly distributed in the cell (this makes it take time ) and everyone has different concentration, when molecule’s concentration be higher the reaction will occur. ❖ Formation of citrate - 3 reactions in this cycle have very high negative ∆G, that what drives the reactions forward. - In the body elsewhere, the reactions of the Krebs cycle are reversible, α-Ketoglutarate to Succinyl CoA is the only irreversible step in the whole reaction cycle. ‫بتسي‬ ‫ لكن يف سلسلة التفاعالت هاي بتكون‬، ‫يعن التفاعالت ممكن تكون منعكسة يف أماكن أخرى من الجسم‬ ‫ر‬ ‫ي‬ ‫وال ما بيكون منعكس أبدا وال يف أي مكان يف الجسم‬ ‫تفاعل‬ ‫ابع‬ ‫ر‬ ‫عدا‬ ‫ما‬ ، ‫بتنعكس‬ ‫وما‬ ‫لألمام‬ ‫ي‬ - ATP-Citrate lyase or ATP-Citratase converts Citrate to acetyl CoA and Oxaloacetate OUTSIDE THE MITOCHONDRIA, not in the cycle. - Other enzymes in the cytosol can convert Oxaloacetate to Malate. ❖ Formation and Oxidation of Isocitrate - Glycolysis is a 10-step reaction that produces pyruvate, which in turn will give acetyl-CoA. - The rate-limiting step is the conversion of fructose6-phosphate, the enzyme catalyzing this reaction is phosphofructokinase. - Phosphofructokinase is regulated by ATP and citrate, so if their concentration get high, they will inhibit phosphofructokinase. THE END OF SHEET #5 6v2 Mera Masalmeh & Farah Saleh Abdullah Mohammed Nafez Abutarboush Krebs cycle (P2) ❖ α-Ketoglutarate to Succinyl CoA As we mentioned in the previous sheet to convert isocitrate into alphaketoglutarate, the process involves oxidizing isocitrate and then releasing a carboxylic group from it (CO2), making this reaction an oxidative decarboxylation reaction. Now, let's detail how alpha-ketoglutarate is converted into succinyl CoA. The alpha-keto dehydrogenase complex is involved in this process, which consists of multiple enzymes, each working in a separate reaction and passing the product to the next enzyme until the final product is produced. IMPORTANT NOTES: → The enzymes in the complex are: 1. Decarboxylase (E1) with thiamine pyrophosphate (TPP) as its coenzyme. 2. Transacylase (E2) with lipoate as its coenzyme, which has a disulfide bond. 3. Dehydrogenase (E3) with FAD as its coenzyme. - α-Ketoglutarate to Succinyl CoA is the only irreversible step in the whole reaction cycle! - CoA from the first Acetyl CoA doesn't enter the cycle (just acetate inters) - Thiamine is vitamin B1 - ΔG for NAD+ (-52.6 Kcal/mol) is greater than FAD (-41 Kcal/mol), that’s why FAD transfer electrons to NAD+ First, TPP from E1 binds to alphaketoglutarate, breaking the bond between the terminal carboxylic group and the keto group, releasing CO2. E1 then releases the remaining alpha-ketoglutarate to get back to its original form, leaving a carbonyl group at the molecule's terminal which is an unstable state. Next, E2's lipoic acid binds to the carbonyl group, which results in breaking to its disulfide bond. E2 then translocate the remaining alpha-ketoglutarate to CoA which stabilizes the molecule and allows its transfer from one place to another. At this point, succinyl CoA is formed. However, E2 has not yet reformed its original structure (the disulfide bond). To do this, E3 with its coenzyme FAD, starts working. FAD accepts electrons from the oxidation reaction to reform the disulfide bond, it then transfers these electrons to NAD+ to complete the process. Note: There are 5 coenzymes involved in the reaction (TPP, Lipoic acid, CoA, FAD, and NAD+), three of them involved within the structure of the enzymes (TPP, Lipoic acid, and FAD). There are other complexes that work in the same mechanism of alpha-ketoglutarate dehydrogenase complex, which are: (Exam question!) 1. Pyruvate dehydrogenase complex: This complex converts pyruvate into Acetyl CoA, CO2, and produces NADH. 2. α-keto acid dehydrogenase complex: This complex acts on the branched-chain amino acids, converting them into their corresponding ketoacids. This process ultimately leads to the production of Acetyl CoA. Remember: Pyruvate composed of 3 carbons, it gives two of them in the form of Acetyl CoA and the one in the form of CO2. All three of these complexes are large enzyme complexes with multiple subunits containing three different enzymes. There is no loss of energy, and the substrates for E2 and E3 remain bound, allowing for a higher rate of reaction. Mutations in the Krebs cycle are rare, but deficiencies in certain parts of it can significantly impact the entire process. As we discussed previously, thiamine is the coenzyme for E1. If a deficiency in thiamine occurs, the enzyme cannot work efficiently, leading to the accumulation of α-ketoglutarate, pyruvate, and α-keto acids (the substrates) in the blood. ❖ OXIDATIVE DECARBOXYLATION OF PYRUVATE: The conversion of pyruvate into acetyl-CoA is resulted by removing of one carbon atom as CO2 and producing of NADH molecule, using Pyruvate dehydrogenase complex (which has a similar mechanism to that of alpha-ketoglutarate dehydrogenase complex and α-keto acid dehydrogenase complex) this mechanism includes producing a substance attached to CoA (this substance will have one carbon less than the substrate) and because pyruvate (the substrate) has 3 carbons, the first step includes removing a CO2 molecule and producing acetyl-CoA (which has 2 carbons) and then oxidation rxn is done by this enzyme to produce NADH. So, these three enzymes have a common product which is NADH, and they will produce a substance attached to CoA After few steps in Krebs cycle as discussed before, now, we have succinyl coA as a product (a 4-carbon molecule) so we need to remove the Coenzyme which resulted in generation of GTP molecule catalyzed by the enzyme succinate thiokinase to produce a succinate. Succinate should be oxidized by Succinate dehydrogenase to produce FADH2, now we are dealing with a double bond in the middle of the carbons which should be hydrated by H2O to produce Malate (the alcohol form of the acid) and it should be oxidized to produce oxaloacetate again from the action of the enzyme Malate dehydrogenase and the 3 rd NADH molecule is produce too. keto acids + α-keto acid dehydrogenase (enzyme) CO2 +NADH + Same substrate without a carboxyl group, coupled to CoA. -This mechanism of pyruvate decarboxylation may have mutations (rarely), or deficiency in one of the coenzymes in the first enzyme of the complex, which causes accumulation of the substrate like pyruvate conversion to acetylCoA, this leads to congenital lactic acidosis. -Mechanism of arsenic poisoning: Arsenic binds covalently to the 2 sulfurs of lipoic acid, breaks the disulfide bond and inhibits its action, thus arsenic is a fatal chemical in high concentrations because it disables Krebs cycle that’s why it is poisoning. It breaks the disulfide bond and attaches itself to the sulfurs. ❖ GENERATION OF GTP: How can we generate ATP? 1. By electrons transport chain (oxidative phosphorylation) which needs O 2. 2. Without O2 (substrate level phosphorylation) Mainly using succinate thiokinase enzyme, this is the reaction we need in Krebs cycle. There are three reactions (substrate level phosphorylation): Present in Glycolysis substrate level phosphorylation → Generating ATP/GTP without needing O2, there are few reactions in our body do that. Succinyl CoA thioester bond, succinate thiokinase, substrate level phosphorylation GTP +ADP ↔ GDP + ATP What applies to ATP applies to GTP ❖ Oxidation of succinate to oxaloacetate: Oxaloacetate is an acid molecule like all compounds in TCA cycle. It is not an enzyme, but it works in the same way as an enzyme- Viewed as a catalyst. It processes the Acetyl CoA and oxaloacetate comes again as unchanged (same idea as an enzyme) An important junction point in metabolism, Citric Acid Cycle, amino acid metabolism (as the corresponding keto acid of aspartic acid) and carbohydrate metabolism Important for gluconeogenesis (we are going to take it in carbs metabolism) Oxaloacetate can’t exit the mitochondria, but malate can do (remember the reversible reaction between them). -Oxidation of succinate to oxaloacetate: ✓ Oxidation of succinate to fumarate by succinate dehydrogenase and FAD. ✓ Hydration of Fumarase, OH- and H+ from water, process fumarate to malate ✓ Alcohol group of malate oxidized to a keto group by malate dehydrogenase and NAD+, give NADH and oxaloacetate. ❖ Bioenergetics of TCA Cycle: - The energy stored in acetate is equal to 228, the overall net ΔG= -228 kcal/mole - The energy stored in the products of the cycle 3 NADH, FAD(H2), and GTP (10ATP) all =207 Kcal/mole To calculate the efficiency of the cycle, we must divide the actual result over the expected one, 207(actual)/228 (expected) = 90%, and this is the most efficient machine in the world. Note: Efficiency can’t be 100% in actual life. -Three reactions have large (-ve) ΔG values, which restricts the movement of the cycle in only one direction. ❖ Net result of the cycle & its significance: Those 10 points are important. Excess carbs turn into fats, how? Carbs like glucose and fructose all go through glycolysis process and at the end produce pyruvate, the pyruvate produces acetyl CoA, which is the substrate for fatty acids production. - Excess carbs turn into acetyl CoA, molecules of acetyl CoA join forming fatty acids. - Fats are burned in the fire of carbohydrates - Fat cannot be converted to glucose because pyruvate dehydrogenase reaction is an absolutely irreversible step Fat → fatty acids → CoA → goes to Krebs cycle → CO2 Note: Acetyl CoA can never be turned into CO2 without the presence of oxaloacetate. ❖ TCA CYCLE INTERMEDIATES: → Intermediates are Precursors for Biosynthetic Pathways - citrate, acetyl CoA, fatty acid synthesis, liver Malate: is a key molecule to gluconeogenesis. - fasting, malate, gluconeogenesis, liver Gluconeogenesis: generation of new glucose from non carbs sources. - Succinyl CoA, heme biosynthesis, bone marrow - α-ketoglutarate, glutamate, GABA, a neurotransmitter, brain - α-ketoglutarate, glutamine, skeletal muscle to other tissues for protein synthesis VERY IMP Glutamate → α-ketoglutarate Glutamate→ neurotransmitter Oxaloacetate → aspartate Pyruvate → alanine Malate → gluconeogenesis ❖ Anaplerotic Routes (Amino Acid Degradation): When we have a deficiency in one of the cycle intermediates, there are some pathways that can synthesize those intermediates by Anaplerotic reactions. ▪ Anaplerotic Reactions: - Pyruvate Carboxylase is a major anaplerotic enzyme (requires biotin) Pyruvate → oxaloacetate - Found in many tissues, liver, kidneys, brain, adipocytes, and fibroblasts. - Very high conc. In liver and kidney (gluconeogenic pathway) why?? Because there is a lot of gluconeogenesis which takes a lot of malate from mitochondria and as the result deficiency of oxaloacetate occurs, so it is found there to maintain the proportion of oxaloacetate. Remember: malate is a substrate for oxaloacetate. - Requires biotin because it is a carboxylation which uses B 7 (biotin). - Acetyl CoA activates Pyruvate Carboxylase because if you have a lot of acetyl CoA you will need a lot of oxaloacetates to bind with it. ❖ REGULATION OF TCA CYCLE: → Have two concept to regulate and determine the rate of the reaction: - (ATP and ADP) and (NADH/NAD+) Ratio, any increase in either one will be coupled to a decrease in the other. - NADH is product of the cycle which come as a product from 3 enzymes, so NADH works as an allosteric inhibitor for these enzymes (when NADH increase it will decrease enzyme’s work (negative feedback). - Correspond to ETC (ATP/ADP) - Two major messengers (feedback): (a) phosphorylation state of adenines (b) the reduction state of NAD - Adenine nucleotides pool and NAD pool are relatively constant. - The slowest step in cycle is conversion of isocitrate to α- ketoglutarate (it is the rate limiting step of the whole pathway). → What happens during it? 1) allosteric inhibitor (NADH) 2) ADP activation (the only step in cycle) 3) Ca+2 activates isocitrate dehydrogenase for cell signaling /muscle movement. - ADP acts as an allosteric activator for only one enzyme in the cycle which is isocitrate dehydrogenase. ❖ Regulation – Citrate & Citrate Synthase: - Rate regulated by oxaloacetate & citrate (inhibitor) - ATP acts as an allosteric inhibitor of citrate synthase. - Effect of citrate: a) Allosterically inhibits PFK, the key enzyme of glycolysis b) Stimulates fructose-1,6-bisphosphatase, a key enzyme of gluconeogenesis c) Activates acetyl CoA carboxylase, a key enzyme of fatty acid synthesis. Isocitrate DH Best regulation (rate-limiting) Allosterically: activated (ADP, Ca+2) Inhibition (NADH) No ADP vs. ADP (KM), a small change in ADP, great effect α- ketogutarate Inhibited: NADH, succinyl CoA, GTP Activated: Ca+2 ❖ Inhibitors of TCA Cycle (Non-Physiological) A. Aconitase (citrate to aconitate) is inhibited by fluoroacetate (noncompetitive inhibition). B. Alpha ketoglutarate dehydrogenase (alpha ketoglutarate to succinyl CoA) is inhibited by Arsenite (noncompetitive inhibition). C. Succinate dehydrogenase (succinate to fumarate) is inhibited by malonate (competitive inhibition) The end of sheet #6