Biochem Block 2 PDF

1.Distinguish between catabolism and anabolism. Catabolism - breakdown of macromolecules for energy (ATP, NADH, FADH2, NADPH) Anabolism - synthesis of macromolecules using energy (ATP & NADPH) 2. Understand the importance of electron carriers. Energy is extracted from fuel molecules a...

1.Distinguish between catabolism and anabolism. Catabolism - breakdown of macromolecules for energy (ATP, NADH, FADH2, NADPH) Anabolism - synthesis of macromolecules using energy (ATP & NADPH) 2. Understand the importance of electron carriers. Energy is extracted from fuel molecules as high-potential electrons Oxidation is Loss of electrons (catabolism) Reduction is Gain of electrons (anabolism) 3. Discuss the role of ATP in metabolism. Used to synthesize biomolecules, and maintain homeostasis in the body 4. Distinguish between substrate level and oxidative phosphorylation. Substrate level phosphorylation: when a phosphoryl group is transferred from a substrate to ADP or GDP to form ATP or GTP, coupled with the release of free energy Oxidative phosphorylation: Electrons are passed from one member of the transport chain to another in a series of redox reactions 5. Identify features common to metabolic pathways: substrates, products, enzymes, regulators etc.) and interpret simple metabolic diagrams. 6. Explain common mechanisms of metabolic regulation. Substrate availability Product inhibition Feedback inhibition Feed-forward regulation Allosteric regulation Localization Changes in enzyme activity by covalent modification 7. Discuss the role of metabolism in blood glucose homeostasis. After a meal - BG increases ○ Catabolism: oxidation of dietary glucose for energy ○ Anabolism: synthesis of high energy storage molecules (fats/glycogen) using excess glucose During fasting/exercise - BG levels are lower & must be maintained ○ Catabolism: breakdown of energy storage molecules (glycogen/fat), proteins and oxidation of fatty acids ○ Anabolism: synthesis of endogenous glucose & ketone bodies as an alternate fuel 8. Discuss the regulation of metabolism by energy levels and calcium. Ca2+ is released from the ER, it activates many enzymes of catabolic pathways ○ Muscle (exercise) and liver (BG) 1. Explain the importance of cell-cell communication in the regulation of metabolism. Regulation of cellular processes (metabolism and gene expression) Cells respond to environmental signals by regulating activities 2. List the 4 different types of receptors and their basic mechanism of action. GCPR - activation leads to the production of second messengers Gated ion - binding of ligand opens or closes ion channel  Steroid - binding of steroid hormones act as a ligand-activated transcription factor. This alters the rate of gene expression Receptor - catalyzes the production of intracellular signaling molecules 3. Give examples of the four types of receptors. GPCR - epinephrine/norepinephrine (beta / alpha adrenergic) Gated Ion - nictonic, acetylcholine, gaba, glycine Steroid - androgen, progesterone, estrogen Receptor - insulin, tyrosine kinase 5. Indicate the receptors, G-protein and effector enzyme in each of these systems. Adenylate cyclase ○ Receptors: b-adrenergic or glucagon ○ G-protein: Gsa ○ Effector: adenylate cyclase converts atp to cAMP Phosphoinositide second messenger system ○ Receptors: a-adrenergic ○ G-protein: Gqa ○ Effector: phospholipase C 6. Indicate what second messenger(s) are produced by activation of adenylate cyclase and phospholipase C. Adenylate cyclase: ATP to cAMP Phospholipase C: PIP2 to IP3 and DAG & also Ca2+ 7. Indicate which protein kinase (PKA, PKC) is activated in both systems Adenylate cyclase: PKA Phosphoinositide: PKC 8. Learn mechanisms of termination of the signaling effect Removal of ligand GTPase deactivates receptor (GTP->GDP) Degrade second messenger Phosphatases dephosphorylate 1. Describe Gibbs free energy and how it is related to enthalpy and entropy Max amount of useful energy available to do work Combines enthalpy and entropy into a single value. The change in free energy, ΔG, is equal to the sum of the enthalpy plus the product of the temperature and entropy of the system 2. Review the major laws of thermodynamics 1: energy can neither be created nor destroyed, only changed from one form to another 2: every energy transfer or spontaneous transformation increases the entropy (disorder) of the universe 3. Relate the standard free energy change to the Keq under standard conditions 4. Define and differentiate between ΔG, ΔG°, and ΔG°` ΔG - predicts direction of reaction toward equilibrium ΔG° - free energy change under standard conditions (1M, 298K, 1atm) ΔG°` - free energy change under physiological conditions (all above and ph 7) 5. Explain the difference between an endergonic reaction and an exergonic reaction 6. Describe how to calculate the actual ΔG of a cellular reaction if you are given the ΔG°` and the cellular concentrations in [M]. 7. Describe the difference between super-high energy compounds, high energy compounds, and low energy compounds Super high High: free energy of hydrolysis equal to or higher than ATP, negative delta G low 8. Describe the various bonding arrangements in ATP, and define the α, β & γ – phosphate High energy phosphate bonds due to large electrostatic repulsive forces The three phosphate groups, in order of closest to furthest from the ribose sugar, are labeled alpha, beta, and gamma 9. Learn the concept of substrate level phosphorylation when a phosphoryl group is transferred from a substrate to ADP or GDP to form ATP or GTP, coupled with the release of free energy 10. Be able to calculate the ΔG of a coupled reaction if given the ΔG values of the individual reactions 1. Identify the major GLUT transporters and the tissues where they are found. GLUT-1: in most tissues (brain/erythrocytes) - basal glucose uptake - km = 1 GLUT-2: liver, kidneys, pancreas - removes excess glucose from blood (high km) - km = 15-20 GLUT-3: most tissues (neurons, placenta, testes) - basal glucose uptake - km = 1 GLUT-4: muscle and fat - insulin-dependent - km = 5 2. Explain the purpose of glycolysis in the cell, its cellular location and list the rate limiting enzymes. Oxidation of glucose to 2 molecules of pyruvate Location: cytoplasm Rate limiting enzymes ○ hexokinase ○ Phosphofructokinase 1 ○ Pyruvate kinase 3. Describe the key reactions of glycolysis. Note the reversible and irreversible steps. 3 major stages: ○ Energy investment ○ Cleavage ○ Energy generation 3 irreversible rxns: ○ glucokinase/hexokinase ○ phosphofructokinase -1  ○ Pyruvate kinase 2 substrate-level phosphorylation reactions synthesize ATP: ○ Phosphoglycerate kinase ○ Pyruvate kinase 4. Indicate the total energy output of glycolysis under aerobic and anaerobic conditions. aerobic glycolysis of glucose to pyruvate can produce 6-8 molecules of ATP as opposed to 2-3 molecules of ATP for anaerobic glycolysis 5. Compare and contrast the tissue specificity of Glucokinase and hexokinase, their kinetic parameters and their respective regulation. Glucokinase is present only in the liver while hexokinase is present in all tissues of the body including the liver. 6. Explain the regulation of PFK-1 and Pyruvate kinase and correlate activators and inhibitors of the reactions with the flow of metabolism. PFK-1 is tightly controlled by the ATP of the cell ○ Activators: F2,6BP (allosteric activator) ○ Inhibitors: citrate (too much will build up in cytosol) Pyruvate kinase ○ Activators: F1,6BP (feed-forward activation) Activating PFK-1 increasing production of F1,6BP and activity of PK ○ Inhibitors: acetyl coA At high energy levels, glycolysis is inhibited by ATP-citrate lyase 7. Distinguish between aerobic and anaerobic glycolysis and the role of NADH in anaerobic glycolysis. 8. Explain the outcome of pyruvate kinase deficiency. Hemolytic anemia - decreased ATP and lactate production 9. Identify far from equilibrium reactions (often called irreversible) and near equilibrium reactions (often called reversible). FAR FROM = IRREVERSIBLE NEAR EQUILIBRIUM = REVERSIBLE 1. Indicate how pyruvate is transported from the cytosol into the mitochondrial matrix Pyruvate transporter & protons (H+) 2. Summarize the overall reaction catalyzed by the PDH complex. Oxidizes pyruvate to acetyl coA Links citric acid cycle to glycolysis Oxidative decarboxylation 3. Outline the cofactors that are required by the PDH complex (TPP, lipoic acid, FAD, NAD, coenzyme A) TPP Lipoic acid FAD NAD Coenzyme A 4. Outline the regulation of the PDH complex  Feedback inhibited by: ○ High energy levels ○ NADH Activated by: ○ Pyruvate ○ Ca2+ 5. Discuss the consequences of thiamine deficiency (Wernicke-Korsakoff syndrome, BeriBeri), and its effect on PDH and α-ketoglutarate dehydrogenase complexes. Lack of thymine, cannot convert sugars to energy (or as much energy that is needed) Leads to heart failure and increased cardiac output, decreased ATP 6. Explain the central role of the TCA cycle in metabolism, including both its catabolic and anabolic functions. Oxidizes acetyl coA to carbon dioxide Generates reduced enzymes NADH and FADH2 Provides biosynthetic precursors for amino acids, nucleotides, fatty acids and glucose biosynthesis 7. Outline the intermediates and the enzymes of the TCA cycle and CO2- producing steps. Intermediates: citrate, isocitrate, a-ketoglutarate, succinyl coA, succinate, fumarate, malate, oxaloacetate Enzymes: citrate synthase, aconitase, isocitrate DH, a-ketoglutarate DH, succinyl coA synthetase, succinate DH, fumarase, malate DH CO2 producing steps: ○ Isocitrate -> a-ketoglutarate via isocitrate DH ○ A-ketoglutarate -> succinyl coA via a-ketoglutarate DH 8. Explain the energetics of the cycle, including reactions where, reducing equivalents (NADH and FADH2 ) are produced, and the production of GTP by substrate level phosphorylation. NADH produced: ○ Isocitrate -> a-ketoglutarate via isocitrate DH ○ A-ketoglutarate -> succinyl coA via a-ketoglutarate DH ○ Malate -> oxaloacetate via malata DH FADH2 produced: ○ Succinate -> fumarate via succinate DH 9. Outline the regulatory mechanisms for the TCA cycle. Indicate the effect of NADH, ATP, Ca2+, and oxaloacetate. 1. Define gluconeogenesis and identify tissues where it occurs. Glucose synthesized from non-carb precursors and is released into the blood Occurs in the liver (90%) and kidney (renal medulla - 10%) 2. Identify the physiological conditions when gluconeogenesis is most active. Most active with low glucose levels which stimulate glucagon (glucagon stimulates gluconeogenesis) 3. List the major gluconeogenic precursors and the tissues that supply each precursor. Pyruvate mostly formed from:  ○ Alanine: transaminated to pyruvate by alanine aminotransferase; released by muscle taken up by liver ○ Lactate oxidized to pyruvate by lactate DH; in muscle Glycerol also a major precursor, from TAG hydrolysis in adipose tissues 4. Identify the conditions that favor gluconeogenesis from each precursor. 5. List the tissues that can provide the bloodstream with glucose. Liver kidneys 6. List the tissues that rely on glucose during fasting and stress. Brain RBCs 7. Identify the four enzymes that catalyze the irreversible steps of gluconeogenesis. Pyruvate carboxylase PEP Carboxykinase F-1,6 bisphosphatase 8. Outline the amount of ATP required to synthesize a molecule of glucose. 4 ATP, 2 GTP, and 2 NADH are used. 9. Describe the regulation of gluconeogenesis: allosteric and hormonal. Allosteric: ○ Acetyl coA activates gluconeogenesis (inhibits PDH) ○ AMP inhibits Fructose 1,6BPase ○ F2,6BP inhibits F1,6BPhosphatase ○ ATP activates Fructose 1,6 bisphosphatase Hormonal: ○ Insulin inhibits Glucose-6-phosphatase Fructose-6-phosphatase PEP-CK ○ Glucagon activates Glucose-6-phosphatase Fructose 1,6 bisphosphatase PEP-CK 10. Discuss the thermodynamic and kinetic constraints of gluconeogenesis. 1. Describe the role of the inner mitochondrial membrane in electron transport and oxidative phosphorylation. 2. Describe the path electrons flow from NADH and FADH2 to oxygen, through the ETC complexes (I through IV). Complex I (NADH DH): uses FMN as a cofactor Complex II (Succinate DH): uses FAD as a cofactor  Complex III (cytochrome reductase or cyt bc1) uses heme group (iron) as cofactor Complex IV (cytochrome oxidase or cyt a,a3) uses copper (Cu2+) & heme groups as a cofactor Path: ○ 1. Complexes I and II donate electrons to CoQ ○ 2. CoQ donates electrons to complex III ○ 3. Complex III donates electrons to CytC ○ 4. CytC donates electrons to complex IV ○ 5. Complex IV donates electrons to oxygen ○ 6. Oxygen reduced to water 3. Outline the different types of electron carriers found in the ETC complexes Electron carriers: ○ CoQ Lipid soluble, in inner mitochondria Carries electrons from I to III and II to III ○ Cyt C Hydrophilic in intermembrane space Carries electrons from III to IV 4. Explain how the electrochemical gradient is generated during electron transport 5. Describe ATP synthesis with reference to Mitchell’s Chemiosmotic theory. 6. Discuss the mode of action of specific inhibitors of the ETC Atractyloside ○ Binds outward facing (intermembrane space) portion of the adenine nucleotide transporter Bongkrekic acid: ○ Binds inward facing (matrix) portion of adenine nucleotide transporter 7. Define and differentiate between inhibitors of the ETC and uncouplers of oxidative phosphorylation. Inhibitors: block electron transfer through the complexes ○ Decrease ATP synthesis ○ Decrease ETC and oxygen consumption Couplers: destroy the electrochemical gradient (creates leaks in membrane) ○ Decrease ATP synthesis ○ Increase ETC and oxygen consumption BOTH decrease ATP synthesis 8. Define uncoupling of oxidative phosphorylation with reference to the different types of uncouplers and outline the role of natural uncoupling in brown adipose tissue. Destroy electrochemical gradient ○ Effects are decrease in ATP synthesis and increase in ETC and O2 consumption Thermogenin: in brown adipose tissue ○ The H+ gradient generated from electron transport is uncoupled from ATP synthesis and generates heat  Ex brown bears in hibernation: active ETC provides water, uncoupling provides heat 2,4 dinitrophenol: lipophilic proton carrier; generates heat, no protein gradient so no ATP synthesis Ionophores: channel formers or mobile carriers ○ Gramicidin, Valinomycin 9. Discuss respiratory control of the ETC and the P/O ratio. Ratio of ATP formed per O atom reduced 3 ATP:1NADH 2ATP:1FADH2 (less bc it bypasses complex I of ETC 10. Outline the aspartate-malate (humans) and glycerophosphate (primarily white muscle tissue) shuttles and their significance in regenerating cytoplasmic NAD+. Glycerophosphate: synthesis of 2 ATPs per systolic NADH oxidized ○ Lose some energy Malate-aspartate: synthesis of 3 ATP per systolic NADH oxidized ○ Doesnt lose energy