Lecture 8 - Digestion, Metabolism, Carbohydrates (PDF)

# Lecture 8 - Digestion, Metabolism, Carbohydrates ## 1. ATP and its functions **Adenosine triphosphate (ATP):** cellular currency derived from energy sources (fuel/light) - Hydrolysis highly exergonic due to the presence of unstable bonds **Energy used for:** - Mechanical work - Active transpo...

# Lecture 8 - Digestion, Metabolism, Carbohydrates ## 1. ATP and its functions **Adenosine triphosphate (ATP):** cellular currency derived from energy sources (fuel/light) - Hydrolysis highly exergonic due to the presence of unstable bonds **Energy used for:** - Mechanical work - Active transport of molecules/ions - Synthesis of macromolecules - Unfavorable reaction coupling **Structure:** - Adenine - Ribose - 3 x Phosphate group ## 2. Oxidation-reduction reactions - **Oxidation reaction:** lose electrons, hydrogen, or gain oxygen - **Reduction reaction:** gain electrons, hydrogen, or lose oxygen ## 3. Carrier Molecules - **Carrier molecules:** molecules used in multiple metabolic reactions to carry a molecular group or electrons - **ATP:** activated carrier of phosphoryl groups - **NAD+:** nicotinamide adenine dinucleotide (activated carrier of electrons) - **FAD:** flavin adenine dinucleotide (activated carrier of electrons) - **Coenzyme A:** activated carrier of acyl groups (e.g., acetyl group) ## 4. Entry of saccharides into the glycolytic pathway **Important Enzymes:** - **Alpha-amylase:** cleaves α1→4 glycosidic bonds in starch (present in saliva) - **Maltase:** cleaves maltose into glucose - **Glycogen phosphorylase:** phosphorylates glycogen, yielding glucose 1-phosphate - **Phosphoglucomutase:** moves the phosphate group from C-1 to C-6, forming G6P. - **Galactokinase:** uses ATP to phosphorylate galactose at C-1, forming galactose 1-phosphate. - **G1P uridyltransferase:** uses UDP-glucose to convert galactose 1-phosphate to glucose 1-phosphate (G1P). - **Phosphoglucomutase:** moves the G1P phosphate group from C-1 to C-6, forming G6P. - **Fructokinase:** phosphorylates fructose at C-1, forming fructose 1-phosphate (F1P). - **Aldolase:** cleaves F1P into DHAP and glyceraldehyde (GA). - **Triose kinase:** phosphorylates GA at C-3, forming G3P. **Polysaccharides:** - **Starch:** plants' form of stored glucose - Contains two types of glucose polymers: amylose and amylopectin - **Glycogen:** storage form of glucose in animal cells - Polymer of α1→4 linked glucose subunits - α1→6 linked branch points occur every 8-12 residues - **Cellulose:** linear, unbranched only in plants - Linked by β1→4 glycosidic bonds - **Chitin:** N-acetyl glucosamine residues in β1→4 linkage - Second most abundant polysac. - Non-digestible by humans but possible in other mammals - Forms part of fungi cell wall - **Heteropolymer**: different monosaccharides included. - **Glycosaminoglycans:** linear polymers of repeating disaccharide units - One will have a negative charge - One always N-acetylglucosamine or N-acetylgalactosamine - **Glycoproteins:** oligosaccharides covalently bound to protein - Present on outer surface of PM, blood, and organelles, and present in ECM - Differ in types of attachments: - **O-linked:** glycoside bond between carbohydrate and OH of Ser/Thr residue - **N-linked:** N-glycosyl bond between carbohydrate and amide of Asn residue - **Erythropoietin:** glycoprotein secreted by kidney into blood to stimulate RBC production - **Glycolipids:** plasma membrane components where oligosaccharides act as hydrophilic head groups - **Glycosphingolipids:** contain a specific backbone structure that acts as signaling molecules in cells - **Gangliosides:** membrane lipids in euk. cells where oligosaccharides containing sialic acid and other monosaccharides act as polar head group - **Lipopolysaccharides:** present on the surface of bacteria # Lecture 9 - Glycolysis - **Glycolysis:** nearly universal pathway for producing ATP by oxidizing glucose. - **Location:** cytoplasm **General Process:** 1. **Preparatory Phase:** - Requires 2 ATP - Glucose is converted to fructose 1,6-bisphosphate - Bond between C-3 and C-4 is broken to yield 2 trioses. 2. **Payoff Phase:** - Produces 4 ATP, 2 NADH, & 2 pyruvate (triose). - Both sugars oxidized at C-1. - Energy from oxidation conserved as NADH and ATP. **Overall Reaction Equation:** glucose + 2NAD+ + 2ADP + 2Pi → 2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O • **Free Energy Change ΔG´°:** -85 kJ/mol (essentially irreversible) ### **Enzymes of Glycolysis, & their function:** - **Hexokinase:** phosphorylates C-6, yielding glucose 6-phosphate (G6P) - Allosterically inhibited by G6P - Traps glucose in the cell - Largely irreversible - **Phosphoglucose isomerase:** converts G6P to fructose 6-phosphate (F6P) - Reversible reaction - **Phosphofructokinase (PFK):** phosphorylates C1, yielding fructose 1,6-bisphosphate(F16P) - Traps sugar as fructose - Irreversible - Allosterically regulated: - **ATP:** inhibitor - **Citrate:** inhibitor - **AMP:** activator - Rate-limiting step of glycolysis - **Aldolase:** cleaves F16P at C-3/C-4 bond, yielding glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) - Reversible - **Triose phosphate isomerase:** interconverts G3P and DHAP - Reversible - **Glyceraldehyde 3-phosphate dehydrogenase (G3PDH):** oxidizes G3P and adds phosphate yielding 1,3-bisphosphoryglycerate (1,3-BPG) - Reversible - Occurs in two steps - **Exergonic oxidation of C1 in G3P and reduction of NAD+ to NADH** - **Endergonic addition of phosphate, forming 1,3-bisphosphoglycerate** - **Phosphoglycerate kinase:** transfers the phosphate group from 1,3-BPG to ADP, forming ATP and 3-phosphoglycerate (3-PG) - Reversible - **Phosphoglycerate mutase:** shifts the phosphate group from C-2 to C-3 of glycerate forming 2-phosphoglycerate (2-PG). - Reversible - **Enolase:** removes H2O molecule from 2-PG, producing phosphoenolpyruvate (PEP) - Reversible - PEP has high phosphate-transfer potential - **Pyruvate kinase:** transfers the phosphate group from PEP to ADP, yielding ATP and pyruvate - Irreversible - Highly exergonic ## Regulation of the Glycolytic Pathway: - **Hexokinase:** allosterically inhibited by G6P (its product) - **Phosphofructokinase (PFK):** allosterically inhibited by ATP, allosterically activated by AMP: - ATP presence lowers affinity for F6P - AMP competes with ATP for the allosteric site - **Pyruvate kinase:** allosterically inhibited by ATP and alanine, stimulated by F16P (much earlier intermediate) - Example of feedforward activation ## 11. Phosphoryl-Transfer Potential - **Phosphoryl-transfer potential:** standard free energy involved in the hydrolysis of a phosphoryl containing compound - Used to compare the tendency of organic molecules to transfer the phosphate group to an acceptor molecule. - ATP has an intermediate level phosphate-transfer potential among the important phosphate molecules. ## 12. Substrate-Level Phosphorylation - **Substrate-level phosphorylation:** formation of ATP via transfer of a phosphate group from a substrate to ADP. - **Doesn't require oxygen** - Only source of ATP production in anaerobic conditions/organisms ### **Enzymes that carry out Substrate-Level Phosphorylation in Glycolysis:** - **Phosphoglycerate kinase:** transfers the phosphate group from 1,3-BPG to ADP, forming ATP and 3-phosphoglycerate (3-PG) - Reversible - **Pyruvate kinase:** transfers the phosphate group from PEP to ADP, yielding ATP and pyruvate. - Irreversible - Highly exergonic ### **Enzymes that carry out Substrate-Level Phosphorylation in the TCA Cycle:** - **Succinyl-CoA synthetase (ligase):** cleaves the thioester bond of succinyl-CoA, forming succinate. - Regenerates CoA - Intermediate, succinyl phosphate, has high phosphate-transfer potential - Donates the phosphate group to ADP/GDP - Forms ATP/GTP via substrate-level phosphorylation. # Lecture 10 - TCA Cycle - **TCA Cycle:** harvests electrons from carbon fuels. - **Location:** mitochondrial matrix **Overall Reaction Equation:** 2 acetyl CoA + 6 NAD+ + 2 FAD + 2ADP + 2Pi → 4 CO2 + 6 NADH + 6 H+ + 2 FADH2 + 2 ATP ### **Enzymes of the TCA Cycle:** - **Citrate synthase (ligase):** joins acetyl CoA with oxaloacetate via condensation, forming citrate. - Irreversible - CoA regenerated - Exhibits induced fit - **Aconitase (isomerase):** rearranges citrate, forming isocitrate. - Reversible - Endergonic, requires flux through the pathway to pull the reaction forward - **Isocitrate dehydrogenase:** converts isocitrate to α-ketoglutarate via oxidative decarboxylation (CO2 released). - Irreversible - Reduces NAD+ to NADH - **α-ketoglutarate dehydrogenase complex:** converts α-ketoglutarate to succinyl-CoA via oxidative decarboxylation (CO2 released). - Irreversible - Reduces NAD+ to NADH - **Succinyl-CoA synthetase (ligase):** cleaves the thioester bond of succinyl-CoA, forming succinate - Regenerates CoA - Intermediate, succinyl phosphate, has high phosphate transfer potential - Donates the phosphate group to ADP/GDP - Forms ATP/GTP via substrate-level phosphorylation - **Succinate dehydrogenase:** oxidizes succinate to fumarate and reduces FAD to FADH2 - Reversible - Flavoprotein present within the IMM - Part of ETC Complex II - FADH2 generated never leaves complex, instantly sent down ETC - **Fumarase (lyase):** converts fumarate to L-malate via hydration - Reversible - **L-malate dehydrogenase:** oxidizes malate to oxaloacetate and reduces NAD+ to NADH - Reversible ## 13. Regulation of the TCA Cycle: - **Pyruvate dehydrogenase** - Activated by: low ATP/ADP ratio, lack of acetyl CoA - Inhibited by: high ATP/ADP and NADH/NAD+ ratios, ample fatty acids/acetyl CoA available - **PFK** - Negatively regulated by citrate. ## 14. ATP Production from NADH/FADH2 - ATP generated from NADH versus FADH2 - The number of ATP generated from one glucose. | Form of Energy | ATP Conversion (ATP) | ATP produced (Pyruvate) | ATP produced (Glucose) | | ------------- |-------------| ------------- | ------------- | | ATP | 1 | 1 | 1 | | NADH | 2.5 | 7.5 | 15 | | FADH2 | 1.5 | 1.5 | 3 | | **Total** | | 10 | 20 | - **Anaplerotic reaction:** replenish intermediates of the TCA cycle. - **Example**: pyruvate carboxylase: catalyzes carboxylation of pyruvate, forming oxaloacetate - **Location:** kidney, liver, brown adipose tissue. - **Function:** Utilize ATP allowing the TCA cycle to continue. # Lecture 11 - Ox Phos: Electron Transport Chain - **Electron Transport Chain** - Location: Inner Mitochondrial Membrane (IMM), the location where ATP is synthesised - The chain generates a proton gradient across the IMM, which is used to drive ATP synthesis. - **Key Reactants and Products:** - **NADH:** - Generated by glycolysis, the pyruvate dehydrogenase reaction, and the TCA cycle. - Must be transported into the IMM using one of two shuttle systems. - **FADH2:** - Generated within Complex II of the ETC. - **Oxygen (O2):** - The final electron acceptor in the ETC. - Reduced to water (H2O). - **ATP::** - Synthesised by ATP synthase. ### Key Components of the Electron Transport Chain - **Complex I (NADH:Q oxidoreductase)** - Pumps 4H+ per NADH - Electron path: NADH → FMN → Fe-S → Q - Q (Ubiquinone) is reduced to QH2 and enters the Q pool. - **Complex II (Succinate-Coenzyme Q reductase)**: - Pumps 0H+ - Electron path: FADH2 → Fe-S → Q - Q is reduced to QH2 and enters the Q pool. - **Complex III (Q-cyt c oxidoreductase)**: - Pumps 4H+ per QH2 - Electron path: QH2 → cyt b → Fe-S → cyt c - **Complex IV (cyt c oxidase):** - Pumps 2H+ per 2 cyt c (4H+ pumped from the matrix) - Used to reduce O2 to 2H2O. - Electron path: cyt c Cu → O2 - **Ubiquinone (Coenzyme Q10 (Q):** - Transfers two electrons at a time - Contains long hydrophobic side chains - Allows for mobility within the inner mitochondrial membrane - Accepts one electron at a time, allowing for several oxidation states. - **Cytochromes:** - Transport one electron at a time - Synthesised within the mitochondria - Contain heme prosthetic groups - **Cytochrome c (cyt c):** - Mobile carrier - Moves within the intermembrane space - Loosely associated with the IMM - **Q cycle:** - Couples the transfer of electrons from QH2 to cytochrome c - Steps from the fact that QH2 carries two electrons while cyt c carries only one - 2 cyt c proteins are reduced for every QH2 oxidized - 4 protons are pumped into the intermembrane space - 2 protons removed from the matrix - 2 protons taken from QH2 ## 15. ATP Synthase - **ATP synthase:** also known as the F1F0 ATP synthase, is a complex enzyme that uses the proton gradient across the IMM to synthesis ATP. - **Fo subunit (membrane-embedded portion):** - Site of protein translocation - **a subunit:** stationary membrane protein that protons pass through. - **c ring:** rotates as protons move down their gradient, driving conformational changes in F1. - Rotation driven by transient protonation on glutamate resides in each c-subunit - **b subunit:** forms a column that connects the Fo subunit to the F1 subunit - **F1 subunit (catalytic, water-soluble portion):** - **Central stalk:** runs up the middle of the hexameric ring, into the c-ring - **Hexameric Ring:** portion of the enzyme that combines ADP and Pi to form ATP. - Composed of 3 α-subunits and 3 β-subunits - **β subunits:** - Each contain an active site that can exist in three different conformations - Shift in conformations driven by a 120° CCW rotation of the central stalk (γ-subunit) - **O (open, empty):** nucleotides free to leave/enter the active site - **L (loose, ADP):** nucleotides are trapped in the active site - **T (tight, ATP):** the phosphate group is forcibly attached to ADP, forming ATP ### **Mechanism of ATP Synthesis** 1. **Proton enters the intermembrane space.** 2. **Proton enters the half-channel facing the intermembrane space** 3. **Binds to a glutamate residue on a c-ring subunit** 4. **After a full rotation of the c-ring, it enters the half-channel facing the matrix.** 5. **Here it is free to move into the matrix, down the proton gradient.** ### **Regulation of ATP Synthesis:** - **Mass-action ratio or energy charge ratio ([ATP]/([ADP][P]1):** - Regulates respiration - The ratio is typically very high but when lowered, the rate of respiration increases. - **Acceptor control:** ADP regulates oxidative phosphorylation. ## 16. Uncouplers and Inhibitors of ETC and ATP Synthesis : - **Uncouplers:** - Increase the permeability of the IMM to protons - Dissipate the proton gradient. - Decrease ATP synthesis. - **Inhibitors:** - Block the flow of electrons through the ETC. - Interfere with ATP synthesis. # Lecture 12 - Ox Phos: ATP Synthesis - **Chemiosmotic theory:** - The ETC and ATP synthesis are coupled by a proton gradient across the IMM. - Explanation: - The energy stored in the electrochemical gradient created by the ETC is used to produce ATP. - Protons are pumped from the matrix into the intermembrane space - This creates a higher [H+] in the intermembrane space and a lower [H+] in the matrix. - The potential difference between the two compartments is called the Proton-motive force (PMF). ## 17. Free energy change, reduction potential, and the redox reactions - **Free energy change (ΔG´°)**: - Indicates how likely a redox reaction is to occur - **Reduction potential (E0):** - Measures a molecule's electron-transfer potential - **Negative E0:** A strong reducing agent (readily donates electrons). - Reductant is oxidized when it donates electrons - **Positive E0:** Strong oxidizing agent (readily accepts electrons). - Oxidant is reduced when it accepts electrons - **Electron transfer:** - Occurs from positive to negative. ## 18. The Electron Transport Chain - **Electron Transport Chain:** - A set of oxidation-reduction reactions that generate a proton gradient used to power ATP synthesis. - Consists of four large complexes within the IMM. - **Complex I (NADH- Q oxidoreductase):** - Pumps 4 protons per NADH - Consists of several subunits, including FMN, iron–sulfur clusters, and ubiquinone. - **Complex II (Succinate-Coenzyme Q reductase):** - Pumps 0 protons per FADH2 - Acts as a conduit for electrons from FADH2 - **Complex III (Q-cytochrome c oxidoreductase):** - Pumps 4 protons per QH2 - Requires the movement of electrons from QH2 to cytochrome c. - **Complex IV (cytochrome c oxidase):** - Pumps 2 protons per 2 cyt c (4 protons pumped from matrix). - Uses heme groups to reduce O2 to H2O. - **Mobile Electron Carriers** - **Ubiquinone (Coenzyme Q10 (Q):** - Transfers two electrons at a time - Moves between the four complexes of the ETC. - **Cytochrome c:** - A small, soluble protein associated with the inner mitochondrial membrane - Carries one electron at a time. ## 19. Reactive Oxygen Species (ROS) - **Reactive Oxygen Species (ROS):** - Oxygen-containing compounds that react with and damage cellular biomolecules. - Contributes to aging and several human diseases. - **Common examples of ROS:** - **Superoxide (O2−):** - Transfer of only one electron to O2 - Product of about 2-4% of oxygen molecules used in the mitochondria. - **Hydrogen peroxide (H2O2):** - Transfer of two electrons to O2 - More damaging to cells than superoxide. - **Hydroxyl Radical (OH·):** - Very reactive. - Likely a reason why the mutation rate of mtDNA is 10X/20X higher than nuclear DNA. - **Protective Enzymes:** - Scavenge ROS to minimize damage - **Superoxide dismutase:** converts superoxide into H2O2. - **Catalase:** cleaves H2O2 into oxygen and water. ## 20. ATP Synthesis - **Proton-motive force:** - The energy stored in the electrochemical gradient across the IMM is used to drive ATP synthesis - The PMF is created by the ETC as protons are pumped from the matrix into the intermembrane space. - **ATP synthase:** - The enzyme that uses the proton gradient to synthesize ATP - A complex, rotary motor protein that is embedded in the IMM. - Consists of two subunits, Fo and F1. - **Fo subunit:** - Embedded in the IMM - Contains a channel through which protons flow from the intermembrane space to the matrix. - **F1 subunit:** - Protrudes into the matrix - Contains the catalytic sites for ATP synthesis. - Has three different conformations, one conformation for each of the 3 subunits. - **Mechanism:** - Protons flow through Fo channel, rotating its c-ring - Rotation of the c-ring causes the rotation of the central stalk (γ subunit). - Rotation of the γ subunit causes the rotation of the F1 subunit. - Rotation of the F1 subunit changes the conformations of the three β subunits. - **Each β subunit undergoes three conformations:** - **Open (O):** - Nucleotides free to leave/enter the active site. - **Loose (L):** - Nucleotides are trapped in the active site - **Tight (T):** - The phosphate group is forcibly attached to ADP forming ATP. - **ATP Synthase is highly regulated:** - **Mass-action ratio or energy charge ratio ([ATP]/([ADP][P]1):** - If this ratio is high, the rate of respiration is decreased because ATP is in high supply. - If the ratio of [ATP]/([ADP][P]1) is low, the rate of respiration increases. - **Acceptor control:** - The rate of ATP synthesis is regulated by the availability of ADP. ## 21. Uncouplers and Inhibitors - **Uncouplers:** - Increase the permeability of the IMM to protons, which dissipates the proton gradient and decreases ATP synthesis: - **Dinitrophenol:** - Very effective uncoupler (also very toxic) - Used to study the chemiosmotic hypothesis - **Inhibitors:** - Block the flow of electrons through the ETC, which stops the production of ATP - **Oligomycin:** - Inhibits ATP synthase - **Cyanide, azide, carbon monoxide (CO):** - Inhibit Complex IV (cytochrome c oxidase). - **Rotenone:** - Inhibits Complex I (NADH:Q oxidoreductase).