BIOCHEM E3 Review PDF

Lecture 1: Metabolism and Energy 1. Metabolic Processes compartmentalized. What are the advantages and disadvantages? The advantages of compartmentalization is that it allows for processes to occur and be regulated efficiently. The disadvantage is that it co...

Lecture 1: Metabolism and Energy 1. Metabolic Processes compartmentalized. What are the advantages and disadvantages? The advantages of compartmentalization is that it allows for processes to occur and be regulated efficiently. The disadvantage is that it costs energy. 2. Structural features of Mitochondria? Contains Outer membrane, Inner Membrane ( cristae) , Intermembrane. 3. What is ATP? Composition, types of phosphate bonds, where is energy stored in ATP? Why does ATP have high phosphoryl transfer potential? ATP is the principal molecule for storing and transferring energy in cells. Contains 1 phosphoester bond and 2 distal phosphoanhydride bonds. ATP has high phosphoryl transfer potential because it has resonance stabilization, electrostatic repulsion, increased entropy, and stabilization due to hydration. 4. ATP Cycle, How is ATP used in metabolic reactions? The ATP cycle goes through the process of hydrolysis when ATP is converted into ADP and energy is released, whereas it goes through condensation when ADP is converted to ATP and energy is consumed. When ATP is converted into ADP a phosphate group is lost and vice versa when ADP is converted to ATP. ATP is used in metabolic reactions because it stores energy for a short term and releases it when a specific process is in need of energy. ATP hydrolysis is an example of a coupled reaction which is unfavorable. 5. Sources of Energy for ATP Synthesis? The sources of energy for ATP synthesis include macronutrients which are fats, carbohydrates, and proteins and micronutrients which include minerals and vitamins. Fats go through the process of beta oxidation to be converted, carbohydrates use glycolysis, and protein use deamination to go through glycolysis to be turned into pyruvate. 6. Difference between Acetyl CoA and ATP? Acetyl CoA and ATP both hold energy in their bonds. Acetyl coa has thioester bonds and ATP has phosphoester bonds, and two distal phosphoanhydride bonds. Acetyl coA is the product of the pyruvate dehydrogenase complex and ATP is the product of the ETC following the The Citric Acid cycle. 7. What are other high-energy molecules? What makes them high energy? Which type of reaction are they used in? How can they be used for synthesis of ATP? Other high-energy molecules include phosphoenolpyruvate (PEP), Creatine Phosphate, Glucose-3-phosphate, and 1,3-Bisphosphoglycerate(1,3 BPG). They can be used for substrate level phosphorylation reactions because they are high energy molecules that can be used to produce ATP, from ADP by phosphorylating it. They are high energy because they have high phosphoryl transfer potential. Energy is only broken off from the distal phosphoanhydride bonds, not the phosphoester bond. 8. Types of Energy Reactions?  Substrate level phosphorylation and oxidative phosphorylation. Substrate level is when high energy substrates are used to generate ATP and oxidative is when a proton gradient generated by electron transport in mitochondria. 9. What is an oxidation-reduction reaction? Its importance? Why are electron carriers needed? How can free radical damage be prevented? Oxidation-reduction reaction is an electron transfer reaction. It is important for the TCA cycle and helps with the entry point. Electron carriers are needed because they are like babysitters and help the electrons get to their appropriate location. Free radical damage can be prevented by electron carriers. 10. Which type of molecules will yield the most energy? Can you identify the most/ least energy-releasing molecule from its structure? The most energy will be yielded by the most reduced form. Methane will be the most energy releasing molecule whereas carbon dioxide will be the least energy releasing as it is most oxidized. 11. Many activated electron carriers are derived from vitamins. NADH, FADH2, CoA, Thiamine pyrophosphate, Biotin, tetrahydrofolate are derived from which vitamins? NADH (niacin) FADH2 ( riboflavin) CoA (pantothenate) Thiamine (thiamine) Biotin ( Biotin B7) Tetrahydrofolate ( Folate) 12. Anabolism vs. catabolism? Anabolism is the synthesis of molecule whereas catabolism is the breakdown of molecules. Anabolism is the energy invested, and catabolism energy is released. 13. Types of chemical reactions: What happens in each of these reactions? Oxidation-Reduction reaction: Electron Transfer. Succinate to Fumarate and also Malate to Oxaloacetate. Ligation Reaction: the formation of covalent bonds ( C-C bonds) Ex. Pyruvate to Oxaloacetate. Isomerization: formation of isomers. Ex. Citrate to Isocitrate Group transfer: transferring a function group from one molecule to another. Ex. Glucose + ATP to Glucose-6- phosphate + ADP. Hydrolysis: Breakdown of bonds with the usage of water. 1 reactants two product Hydration: synthesis of a bond with the usage of water. 2 reactions 1 product. Lyases: cleavage of C-C bonds without hydrolysis or oxidation. Two reactants 1 product. Lecture 2: Metabolism and Energy Contd. 1. Pyruvate Dehydrogenase- Enzyme and cofactors/ coenzymes? Enzymes: E1 ( pyruvate dehydrogenase aka pyruvate decarboxylase), E2 ( dihydrolipoyl transacetylase), E3 ( dihydrolipoyl dehydrogenase) Cofactors: There are five cofactors. Include TPP (E1), CoA,( E2) Lipoate(E2), NAD+(E3), and FAD(E3)  2. Which other enzymes require thiamine? pyruvate dehydrogenase, alpha- ketoglutarate dehydrogenase, and transketolase. 3. What will happen with thiamine deficiency? What substrate will accumulate? Beri Beri (mammillary bodies, colliculi) Wernicke’s Encephalopathy (Acute) Korsakoff Syndrome ( Chronic) Thiamine deficiency = Pyruvate Dehydrogenase or E1 doesn’t work, so you have a buildup of Pyruvate and maybe lactate(?) 4. What product will not be formed? Which tissue will get most affected? Acetyl CoA will not be formed. Brain and nerves (encephalopathy/neuropathy). It will affect the colliculi. 5. What enzymes need lipoic acid (lipoate) for their activity? How does Arsenic and Mercury poisoning affect PDC? ( Role of -SH groups)? E2 ( dihydrolipoyl transacetylase). It will inhibit the PDC because the E2 enzyme will no longer be able to work. The role of the thiol group is that arsenic inhibits the E2 subunit of the PDH complex that requires lipoic enzymes as coenzymes. Arsenic binds to thiol group in the lipoic acid preventing its use for action of the enzyme. 6. What are the 2 substrates required for TCA Cycle? 1. Acetyl CoA and Oxalacetate 7. What is the fate of 2C and 8 electrons in Acetyl- CoA upon entering the TCA cycle? 3 NADH, 1 FADH2, 2CO2, 1 GTP 8. What are anaplerotic reactions? Examples Anaplerotic reactions replenish (fill) the TCA cycle intermediates. All need Co2, Biotin, and ATP (ABC’s). ○ Succinyl CoA: made by converting LCFA to Propionyl CoA. Propionyl CoA Carboxylase converts it to succinyl CoA ○ Oxaloacetate: pyruvate carboxylase turns pyruvate to oxaloacetate Lecture 3: TCA Cycle and The Electron Transport Chain 1. TCA Cycle-how different fuel sources enter the TCA cycle? Fatty acids can enter the TCA cycle by being converted into Acetyl CoA (after undergoing beta oxidation). Amino acids enter the TCA cycle once proteins are deaminated (at various stages: can be turned into pyruvate, can also be synthesized into acetyl coA, or AKG, or Oxaloacetate) 2. TCA cycle-location, Substrates and products Mitochondrial Matirx Substrate: Acetyl CoA and Oxaloacetate Products: 3 NADH, 1 FADH, 1 GTP, 2 CO2 3. Substrate Level Phosphorylation Molecules that have more energy than ATP (PEP, Creatine Phosphate, and 1,3 BPG) can be used to synthesize ATP by phosphorylation of ADP. ○ Oxidative phosphorylation occurs in the mitochondria during ETC.  4. Oxidation reduction reactions of the TCA cycle: Isocitrate – (iso citrate dehydrogenase, then NAD+ to NADH and H+) → oxalosuccinate intermediate – (lose CO2 via Isocitrate dehyd) → AKG (5 carbon) AKG – (NAD+ to NADH, add CoA-SH w AKG dehyd. complex) → Succinyl CoA Succinate – (FAD to FADH2 by Succinate dehydrogenase) → fumarate L-Malate – (NAD to NADH + H via malate dehydrogenase) → oxaloacetate 5. Reaction sites of CO2 release? Synthesis of AKG: from isocitrate to oxalosuccinate…. after oxalosuccinate is formed, isocitrate dehydrogenase takes away a CO2 to form AKG (5 carbons) Taking AKG and turning it into Succinyl CoA using the AKG Dehydrogenation Complex, so you release CO2 when Succinyl CoA is made. 6. Regulatory enzymes, their inhibitiors and activators, Citrate Feedback inhibition? Pyruvate dehydrogenase (inhibited by ATP, acetyl coA and NADH) a-KG Dehydrogenase (ATP, NADH, succinyl CoA) Isocit. dehyd. (inhibited by ATP and NAz[‘DH, activated by ADP, NAD+ and Ca2+) citrate synthase (inhibited by ATP, NADH, Succinyl CoA and citrate) 7. Are all TCA cycle enzymes located in mitochondrial matrix? Which one is the exception; where is it located? Succinate Dehydrogenase is not in mitochondrial matrix. It is located in Inner Mitochondrial. 8. Fluoroacetate poisoning mechanism? Fluoroacetate reacts with citrate to form fluorocitrate, a toxic intermediate. This intermediate binds to aconitase and thus inhibits the TCA cycle. 9. Arsenic poisoning-mechanism? E2 ( dihydrolipoyl transacetylase). It will inhibit the PDC because the E2 enzyme will no longer be able to work. The role of the thiol group is that arsenic inhibits the E2 subunit of the PDH complex that requires lipoic enzymes as coenzymes. Arsenic binds to thiol group in the lipoic acid preventing its use for action of the enzyme. 10. Stereospecific enzyme? Fumarase, because it only synthesizes the L-isomer **** May**** be sterospecific: isocitrate dehydrogenase, by adding it to the other side of the oxalosuccinate after decarboxylation. The Electron Chain 1. What constitutes ETC vs. Oxid Phosphorylation machinery, ETC Location Components, name of complexes, characteristic features of Complex II? The ETC is part of Oxidative phosphorylation machinery. ETC is located in the inner membrane of mitochondria, specifically the cristae. It has 5 complexes: - Complex I: NADH Dehydrogenase - Complex II: Succinate Dehydrogenase - Complex III: Ubiquinol (CoQ)-Cytochrome C reductase (lipid soluble collects electron from complexe 1-2 to 3).  - Complex IV: Cytochrome c Oxidase (COX) ( water soluble and faces intermembrane space). - Complex V: ATP Synthase - Characteristic Feature of Complex II: the only one encoded in the nucleus. Smallest one and only one that does noy pump protons itself. 2. Electron carriers and prosthetic groups, Sequence of Electron flow thru Complexes? Prosthetic groups (redox centers)- Electron transfer potential of NADH and FADh2 is converted to phosphoryl transfer potential of ATP. Protons are pumped at complex 1,3, and 4 Movement of protons generates the electrochemical gradient ( which includes the pH gradient and the voltage gradient, across the inner mitochondrial membrane. 3. O2 has the highest Electron transfer potential. What does it mean? High electron transfer potential means they have a high electron affinity, and that the substrate is also in its oxidized form. O2 has the highest ETP because it is at the end of the ETC, because electrons have already moved (last e- acceptor). 4. CoQ and Q cycle. How many e- does Q receive and how many it passes on? The Ubiquinone (Q) pool is critical to movement of proton shutting from the matrix to intermembrane space for maintenance of the proton gradient or the protomotive force required to generate ATP. Q cycle takes place in complex 2. CoQ carries 2 electrons. It hands over an electron to cytochrome C to pass it on to complex IV. QH2 is formed by taking another electron and a pair of protons from the matrix. 5. Cytochrome C and Complex IV: bimetallic center, how is water released? Positive redox potential of O2 is used to catalyze the reduction of Fe (in heme) and Cu and O2 binds between the two (bimetallic center, peroxide bridges). ○ Two more electrons added from the matrix, and 2 more protons are used to cleave the peroxide bridge. Peroxides → hydroxides. ○ Once peroxides have 2 more protons added, hydroxides → water release 6. How does proton gradient contribute to ATP Production? The proton produced by proton pumping during the ETC is used to synthesize ATP. Protons flow down their concentration gradient into the matrix through the membrane protein ATP synthase, causing it to spin and catalyze conversion of ADP to ATP. 7. ATP synthase: structural components, catalytic unit, F0 subunit, proton channel? Made of F0 (located in the intermembrane space) and F1 (mitochondrial matrix) ○ F1: catalytic activity that actually makes the ATP using Beta subunits. Proton flow turns central g stalk of F1 subunit. Central gamma epsilon stalk connects F0 and F1 together ○ F0: supportive role, provides proton gradient. B2 exterior column (provides structural support similar to Gamma, but no moving F1) ○ Protons passing through H+ channel of subunit = rotation of the G subunit  Proton channel: Blue for C ring and the cytoplasmic half-channel. Inside are negatively charged aspartic acid residues, neutralized by H+ moving through the channel) 8. F1 subunit: types of beta forms, their roles, what moves g subunit, what does this movement do? Can ATP be formed without moving protons? The gamma subunit is the knob that turns and changes the conformation of the 3 beta subunits. ( open form, tight form, and loose form). As you move the knob, ATP is released and makes tight form into loose form. Movement of protons causes release of ATP ( not directly making ATP). ATP release only happens when protons are moved. ATP cannot be formed without the knob moving. 9. ATP-ADP translocase: function? Antiporter whereby ATP is transported out of the mitochondria. Enables the exchange of cytoplasmic ADP for mitochondria ATP. 10. What is respiratory control? Electrons are transferred to O2 only if ADP is concontantly phosphorylated to ATP. More ATP means an increase in oxygen consumption. Last Lecture Questions: 1. Free Radicals- sources, types, Fenton Rx Sources: Endogenous ( oxidative phosphorylation, peroxisomes, and enzymes) Exogenous (chemical-pesticides, drugs/ medications, pollutants, toxins, stress). Types: Molecule Oxygen → Superoxide ion→ hydrogen peroxide → hydroxyl radical → and peroxynitrite. Fenton Reaction: Hydrogen peroxide is converted into fenton reaction. Redox reactive metal ions, such as Fe2+ and Cu1+, catalyze the conversion of H2O2 to hydroxyl radical. Presence of high levels of Free Fe2+ ( hemochromatosis) and Cu1+ ( Wilson’s Disease) can therefore cause serious oxidative damage to cells. 2. Free radical cellular defenses against free radicals. Glutathione peroxidase (GPx) - found in the mitochondria. (water and NAD+) Anti-oxidants (vitamin A/C/E) Catalase - found in the peroxisomes (water and oxygen gas) Superoxide dismutase (2 superoxides → hydrogen peroxide) Minerals - selenium, zinc, manganese (water) 3. Name of enzymes, Rx they catalyze, SOD - clinical condition Glutathione peroxidase (GPx) - in mitochondria. (H peroxide→ water & NAD+) Catalase - found in the peroxisomes (H peroxide → water and oxygen gas) Superoxide dismutase (2 superoxides → hydrogen peroxide) SOD mutation → ALS, excessive free radical damage, DNA/RNA synthesis 4. Name of antioxidants, and their specific role ( if any): Glutathione peroxidase, Thioredoxin Vitamins- Vitamins A,C,E. ( every day, water soluble vitamins) Minerals- Selenium, Zinc, and Manganese ( not everyday, smaller quantities).  5. ETC complex (es) inhibitors, Effect on the ATP production, what products can accumulate for each inhibitor? How would this affect the TCA cycle? Complex I: Rotenone, Amytal Complex II: Carboxin Complex III: Antimycin A Complex IV: Carbon monoxide and Cyanide Complex V: Oligomycin Random: ATP-ADP translocase: Atractylosin is the inhibitor for it. ATP production will go down NADH production will go up because eventually not using the substrate anymore will accumulate substrate The change in NADH would inhibit the TCA cycle. 6. ETC uncoupling - significance. Effect on ATP production? What is produced? What accumulates (if anything)? Effect on TCA cycle? oxidation and phosphorylation being uncoupled would disrupt the proton or electrochemical gradient. Maybe ADP is produced? Definitely ATP is not produced bc there are no protons (or electrochemical gradients) while substrates still undergo oxidation. ○ Possible upregulation of isocitrate dehydrogenase because it is activated by ADP and inactivated by ATP (cant make ATP, cant inhibit → ADP accumulates, can activate the isocit. dehydrogenase). 7. Physiological and non-physiological uncouplers. Physiological uncoupler protein one (UCP1 or thermogenin) ○ Found in brown adipoytes, needed for FA oxidation and heat production Non-physiological uncouplers: ○ Chemicals: 2,4 DNP and pentachlorophenol. ○ Medications: salicylic acids (betahydroxy acid), aspirin