10 - Basics of Enzymes & Regulation of Enzymes.pdf

Basics of Enzymes & Regulation of Enzymes Dr. Sreenilayam Sept. 24, 2024 Reading: Marks’ Basic Medical Biochemistry, 5th Ed. Ch. 8-9 Some slides modified from Farzaneh Dagh...

Basics of Enzymes & Regulation of Enzymes Dr. Sreenilayam Sept. 24, 2024 Reading: Marks’ Basic Medical Biochemistry, 5th Ed. Ch. 8-9 Some slides modified from Farzaneh Daghigh, PhD, Francis Jenney, PhD, and Kimberly Baker, PhD This is the material you are responsible for: Post-transcriptional and Translational Regulation (CBFM) Review of Gene Expression (CBFM) Protein Structure, Folding, and Modification (CBFM) Vitamins (CBFM) Minerals (CBFM) 2  Basics of Enzyme and Enzyme Regulations: Learning Objectives o By the end of this unit, the successful student will be able to do the following, as measured by multiple choice examination: 1. Differentiate between cofactors, coenzymes, and isozymes. 2. Explain the process of substrate molecules being enzymatically transformed into products (including transition state complex and activation energy) and illustrate the process on an energy diagram. 3. Describe lock and key and induced fit. 4. Identify the different functional groups (amino acid side chains, coenzymes, and metal ions) and explain how each contributes to an enzymatic reaction. 5. Describe factors (pH, temperature, concentrations of enzyme and substrate) affecting the rate of a reaction. 6. Explain the importance of a regulatory enzyme in a metabolic pathway using examples. 7. Compare and contrast the mechanisms for regulating enzymes (feedback inhibition, allosteric, hormonal regulation via covalent modification, and induction/repression), and relate to the time required for each. 8. Contrast KD with KM. 9. Compare and contrast between Michaelis-Menten and Lineweaver Burk plots of enzyme kinetics and inhibition, locate K M, Vmax, KM,app, Vi on each plot, and predict the effect(s) of reaction perturbations (i.e. increases in substrate, increases in inhibitor, etc) on each plot. 10. Compare the catalytic mechanism of an allosteric enzyme to an enzyme that follows Michaelis-Menten kinetics.  Basics of Enzymes Covers Learning Objective: 1. Differentiate between cofactors, coenzymes, and isozymes. 2. Explain the process of substrate molecules being enzymatically transformed into products (including transition state complex and activation energy) and illustrate the process on an energy diagram. 3. Describe lock and key and induced fit.  Cofactors vs Coenzymes Cofactor – a component other than the protein portion of an enzyme (i.e. metal ions/minerals, vitamins, GSH, ATP, CoQ, etc.) Coenzyme – organic cofactor that is loosely bound to the apoenzyme and can be easily separated from it (i.e. most vitamins) Prosthetic group – cofactor that is tightly bound to the apoenzyme (i.e. many minerals)  Isozymes Enzymes that differ in amino acid sequence but catalyze the same chemical reaction May have different kinetic parameters, expressed in different tissues, and/or regulated differently Permits the fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage I.e. ALA synthase, heme oxygenase, hexokinase, COX, lactate dehydrogenase (LDH), etc.  Activation Energy and Transition State Activation energy – energy needed for the reactants (substrates) to reach the transition state New bonds are forming and old bonds are breaking (transition state complex) Transition state (point) – molecules have the highest potential energy Can have multiple transition states for coupled (or successive) reactions Endergonic and exergonic only refer to comparison of reactant & products values 7 Marks’ Fig. 8.7  Enzymes (catalysts) lower the activation energy (barrier), thus speeding up a reaction (stabilize the transition state) – typical catalytic power: 106-1014 Does not change the overall G values Would actually see 2 “hills” or “peaks” on the curve with enzyme ATP Reactant molecules Glucose Enzyme Transition state Activation energy (EA) without enzyme 1. E+S ES Free energy (G) Activation energy (EA) 2. ES EP with enzyme Reactants Change in 3. EP E + P free energy (G) Products Progress of an exergonic reaction 8 Copyright © The McGraw-Hill Companies, Inc.  3-D Structure of Enzymes Active site Substrate-binding site Lock and key Induced Fit Transition state complex 9 Marks’ Fig. 8.4  Active Site Site (usually an opening or cleft) where the chemical reaction takes place Contains functional groups that are actively involved in the reaction Coenzymes – enzymes that utilize cofactors (metals or complex organic molecules) as functional groups too Glucose Yeast hexokinase structure 10 Fig. 18.6 Garrett & Grisham 4th Ed.  Substrate Binding & Transition States Substrate binds to the substrate binding site Non-covalent bonds form Amino acids from enzyme or atoms of cofactors Additional bonds form with the enzyme to stabilize the substrate in its transition state Stabilization is how the activation energy is lowered 11 Marks’ Fig. 8.4 Substrate Binding and Specificity Enzymes are highly specific (some more than others) due to chemical shapes and/or interactions within the substrate binding sites 2 mechanisms describe the binding: Lock-and-Key Induced-Fit 12 Marks’ Fig. 8.5  Binding Mechanisms Lock-and-Key Substrate binding site creates a 3-D shape that is complementary to the substrate Non-covalent interactions (hydrophobic, electrostatic, H-bonds, etc.) Induced-Fit Substrate binding induces a conformational change Still must be complementary Functional groups http://www.studyblue.com/notes/note/n/lecture-test-for-packet-3/deck/774786 repositioned to 13 promote reaction  Various Functional Groups in Enzymes Covers Learning Objective: 4. Identify the different functional groups (amino acid side chains, coenzymes, and metal ions) and explain how each contributes to an enzymatic reaction.  Functional Groups Amino acid side chains Ser, Cys, Lys, His: covalent catalysis Polar AAs: nucleophilic catalysis Coenzymes: non-protein organic molecules (vitamins) Activation-transfer - form covalent bond with substrate, then activate it for transfer Redox reactions – accept or donate electrons to activate substrate, but no covalent bond formed Metal ions Electrophiles – electron-attracting groups Substrate binding, stabilizing anions, donate/accept electrons in redox reactions 15  Coenzymes - Biotin Activation-transfer: form covalent bond with substrate, then activate it for transfer Biotin – uses its N to attach to -COO groups in carboxylases Water-soluble vitamin (B7) Covalently linked to Lys 16 Figure 22.2 Garrett & Grisham 4th Ed.  Coenzymes - CoA Activation-transfer: form covalent bond with substrate, then activate it for transfer Coenzyme A – uses its sulfhydryl group to do nucleophilic attacks on carbonyl groups Used in many pathways, but written as CoA and Acetyl-CoA Requires vitamin B5 17 Marks’ Fig. 8.9  Coenzymes - TPP Activation-transfer: form covalent bond with substrate, then activate it for transfer Thiamine pyrophosphate (TPP) – contains carbon with dissociable proton PDH complex -ketogluterate dehydrogenase complex Requires vitamin B1 (thiamine) Marks’ Fig. 8.8 18  Coenzymes - Redox Oxidation-Reduction Oxidized: loss of e- (loss of H or gain of O atoms) Reduction: gain of e- (gain of H or loss of O atoms) Similar to activation-transfer, but no covalent bond formed Functional groups accept or donate electrons I.e. FAD, NAD(P)+ 19 Marks’ Fig. 8.10 Factors That Affect Enzymatic Activity pH Temperature Substrate / Enzyme concentration Michaelis-Menton kinetics Covers Learning Objective: 5. Describe factors (pH, temperature, concentrations of enzyme and substrate) affecting the rate of a reaction.  Affect of pH Enzymes are most active at a certain pH Usually correlates to their cellular location [Life: The Science of Biology, Purves, 4th Edition]  Affect of Temperature Most enzymes function optimally at 37°C Denaturation occurs as temperature increases [Lippincott Biochemistry, 5th Ed., Fig. 5.7] Substrate/Enzyme Concentration Blue – enzyme Red - substrate  Enzyme Regulation and Metabolic Pathways Covers Learning Objectives: 6. Explain the importance of a regulatory enzyme in a metabolic pathway using examples. 7. Compare and contrast the mechanisms for regulating enzymes (feedback inhibition, allosteric, hormonal regulation via covalent modification, and induction/repression), and relate to the time required for each.  Regulation of Enzymatic Reactions Common Types of Enzymatic Regulations Feedback loops (positive or negative) Allosteric regulation (activators or inhibitors) Covalent modification Hormone regulation via covalent modification Induction/repression of genes Figure 17.8, Voet, Voet & Pratt, 3rd Ed.  Feedback Loops A product acts on an upstream enzyme Can be positive or negative feedback Quick form of feedback (seconds) [Cornell, B. 2016. Feedback Inhibition]  Allosteric Regulation The allosteric site is different from the active site A molecule binds to the allosteric site and changes the conformation of the enzyme (i.e. PFK-2; seconds) [OpenStax Biology, Chapter 6.5: Enzymes, Fig. 4]  Hormonal Regulation Via Covalent Modification Hormones bind to receptors and cause activation of kinases or phosphatases, which then regulated enzymes of pathways Cellular response (covalent: sec – min; hormone: min – months) Induction/Repression of Genes [Molec. Biology of the Cell, Alberts, 5th Ed., Fig. 15-6]  Summary of Enzymatic Regulation Time for Regulator event Typical effectors Results change Substrate availability Substrate Change in Seconds velocity Product inhibition Product Change in Vm Seconds and /or Km Allosteric control End product, another Change in Vm Seconds molecule and/or Km Covalent Another enzyme, Change in Vm Seconds to minutes modification hormones and/or Km to days Induction or Hormone, metabolite Change in min to hrs to days Repression the amount of protein  Michaelis-Menten Kinetics Covers Learning Objectives: 8. Contrast KD with KM. 9. Compare and contrast between Michaelis-Menten and Lineweaver Burk plots of enzyme kinetics and inhibition, locate KM, Vmax, KM,app, Vi on each plot, and predict the effect(s) of reaction perturbations (i.e. increases in substrate, increases in inhibitor, etc) on each plot. 10. Compare the catalytic mechanism of an allosteric enzyme to an enzyme that follows Michaelis-Menten kinetics.  Michaelis-Menten Plot Velocity of all enzymatic reactions is dependent on [substrate] Enzyme catalyzed reactions have a substrate saturation E+S ES E+P 32 Marks’ Fig. 9.2 KM and Affinity A smaller KM value indicates a higher affinity for a substrate Half the amount of enzyme will be bound to substrate at the KM value KM values are often in the M or nM range [Lippincott Biochemistry, 5th Ed., Fig. 5.9]  KM vs KD KM - Michaelis-Menten constant KD - Dissociation constant Kinetic constant Thermodynamic constant Measures the impact of substrate True measure of the affinity of concentration on the speed of a a ligand for a binding site of an reaction enzyme Can be used as an indirect i.e.: concentration 50% of measure of affinity in the active ligand will dissociate from site the enzyme Note: doesn’t address speed of the reaction allosteric site Note: for both KM and KD, the smaller allosteric site the number, the bigger the affinity!!  Turnover Number - Examples Turnover number (catalytic constant) kcat = Vmax / [E]total Units = sec-1 Lysozyme takes 2 sec to cleave a glycosidic bond of glycan Catalase – destroys H2O2 molecules! It destroys 40 million H2O2 molecules every single second 35 Garrett & Grisham 4th Ed.  Regulation via Inhibition Inhibitors - decrease the rate of an enzymatic reaction Reversible – diffuse away at a significant rate (i.e. not covalent!) Irreversible – ‘suicide inhibitors’ Covalent (or very, very tight non-covalent) bonds to enzyme Ex: aspirin, penicillin Decreases amount of active enzyme available Transition-state analogs Best inhibitors! Bind more tightly to enzyme than substrate or products Many pharmacologic examples (penicillin, allopurinol, etc) Marks’ Figs. 8.14 & 31.19  Allopurinol: Example of Transition State Analog (low concentration) 37 Marks’ Fig. 8.15  Reversible Inhibition Competitive Inhibitor binds to substrate-binding site (active site) Noncompetitive Inhibitor binds enzyme other than the substrate-binding site (allosteric site) Can be before or after substrate binds Pure: inhibitor binds in a different place than a given substrate (can have 1 or more substrates) Mixed: inhibitor binds outside of substrate- binding sites Uncompetitive Inhibitor only binds the ES complex (not enzyme alone) Only happens in multi-substrate reactions  Lineweaver-Burk Plots Velocity vs [S] gives Taking the inverse of both axes a hyperbolic curve transforms it to a line Easier to make comparisons, determine type of inhibition, etc. Marks’ Figs. 9.2 & 9.3  Competitive Inhibition Vmax is unaffected Vmax definition: … with unlimited substrate What happens to the inhibitor with unlimited substrate added? Km looks different – Km,app (apparent Km) Km,app is increased with inhibitor What does that mean about the affinity for S? 40 Figure 13.13 Garrett & Grisham, 5th Ed  Example – Competitive Inhibition Statin drugs- antihyperlipidemia Lippincott Biochemistry, 5th Ed., Fig. 5.13 Pure Noncompetitive Inhibition Binding of I has no effect on the binding of S for E Decreased Vmax (Vmax’) value than no inhibitor Vmax value Km is the same 42  Mixed Noncompetitive Inhibition Binding of the inhibitor changes the affinity for the substrate Decreased Vmax (Vmax’) value than no inhibitor Vmax value Km,app depends on KI and KI’ values, but differs from Km 43 Figure 13.16 Garrett & Grisham, 5th Examples – Noncompetitive Inhibition Allopurinol (high concentrations) on xanthine oxidase Alanine on pyruvate kinase Uncompetitive Inhibition Inhibitor only binds ES complex Vmax will decrease Some of the ES complex inhibited Km,app decreases Ability to inhibit is dependent on E and S bound Figure 13.17 Garrett & Grisham, 5th Ed  Allosteric Modification Outside of the active site Binding of regulators to allosteric sites induces a conformational change within the enzyme May not follow Michaelis-Menton kinetics (i.e. hexokinase I vs glucokinase) Activators, deactivators, cooperative binding are examples Sigmoidal curve is due to cooperativity 46 Marks’ Fig. 9.4  Cooperativity in Allosteric Enzymes Usually ≥ 2 subunits and ≥ 2 states First substrate (S) has a hard time binding because all subunits in T (taut) state Substrate binding in 1 subunit increases the chance of another substrate binding S-shaped curves Ends in substrate bound to all substrates in the R (relaxed) state I.e. hemoglobin binding O2 47 Marks’ Fig. 9.7  Cooperativity Activators & Inhibitors Activators – increase the affinity of the enzyme for the substrate tend to bind more tightly to R state Inhibitors – decrease the affinity of the enzyme for the substrate tend to bind more tightly to T state Activators shift S-curve to the left Inhibitors shift S-curve to the right Notice the change in affinity values Example: PFK-1 of glycolysis 48 Marks’ Fig. 9.8  Summary of Enzymatic Regulation Time for Regulator event Typical effectors Results change Substrate availability Substrate Change in Seconds velocity Product inhibition Product Change in Vm Seconds and /or Km Allosteric control End product, another Change in Vm Seconds molecule and/or Km Covalent Another enzyme, Change in Vm Seconds to minutes modification hormones and/or Km to days Induction or Hormone, metabolite Change in min to hrs to days Repression the amount of protein  Carbohydrates: Digestion, Absorption and Glycolysis Dr. Sreenilayam Oct. 1, 2024 Reading: Marks’ Basic Medical Biochemistry, 5th Ed. Ch. 21, 22, 27 Some slides modified from Dianzheng Zhang, PhD, Francis Jenney, PhD, and Kimberly Baker, PhD. This is the material you are responsible for: Macronutrients and Healthy Dietary Intake (CBFM) Basics of Hormonal Regulation (CBFM) Basics of Enzymes and Regulation of Enzymes (CBFM) Broad Overview of Metabolism (CBFM) 2 Carbohydrates: Digestion, Absorption & Glycolysis Learning Objectives o By the end of this unit, the successful student will be able to do the following, as measured by multiple choice examination: 1. List and match the nomenclature (i.e. monosaccharides, polysaccharides, pentoses, etc.) and basic structure of carbohydrates, and the idea of enantiomers/isomers 2. Describe the digestion and absorption of the major carbohydrates, including disaccharides and polysaccharides 3. Contrast the roles of the different glucose transporters, their location and identify which are insulin regulated 4. List the 3 steps of glycolysis that are regulated, where they are located and explain how they are regulated 5. Compare and contrast the roles and location of hexokinase and glucokinase 6. Explain the overall stoichiometry of glycolysis 7. Contrast the fate of pyruvate under aerobic vs. anaerobic metabolism and explain why NAD+ must be recycled and how 8. Clearly contrast substrate-level phosphorylation with oxidative phosphorylation 9. Describe how sugars (galactose, lactose, fructose) other than glucose are 3 metabolized  Overview of Carbohydrate Nomenclature & Structures Covers Learning Objective: 1. List and match the nomenclature (i.e. monosaccharides, polysaccharides, pentoses, etc.) and basic structure of carbohydrates, and the idea of enantiomers/isomers  Basic Nomenclature Carbohydrates (“hydrate of carbon”) have empirical formulas of (CH2O)n , where n ≥ 3 Aldehyde Ketone Monosaccharides one monomeric unit Oligosaccharides ~2-20 monosaccharides Polysaccharides > 20 monosaccharides Glycoconjugates linked to proteins or lipids Aldoses - polyhydroxy aldehydes Glyceraldehyde Dihydroxyacetone Ketoses - polyhydroxy ketones (Aldose) (Ketose) The most oxidized carbon: aldoses C-1, ketoses usually C-2 Trioses (3 carbon sugars) are the smallest monosaccharides Pentoses (5 carbon sugars), hexoses (6 carbon sugars) more common Many types of modified sugars (see next slide for examples) 5  Some examples of modified sugars 6 Lehninger 3rd ed.  Isomers & Enantiomers Chiral carbon - carries four different atoms or groups Isomers - molecules have same molecular formula, but different arrangement of the atoms Enantiomers - form non- superimposable mirror images Naturally, most monosaccharides are D-form Conformational isomers - isomers are interconverted just rotating a group on a single bond ( vs ) (α-glucose) (β-glucose)  Formation of Disaccharides anomeric C (α-Glucose) (β-Glucose) Nonreducing end Reducing end (Glycosidic bond) 8 Glc (1→4) Glc Common Dietary Disaccharides Lactose (Milk sugar) Gal (1→4) Glc Sucrose (Table sugar) Glc (1→2) Fruc 9 Polysaccharides 10  Sugars Attached to Proteins & Lipids Glycoproteins – Protein > sugar Membrane bound Secreted Proteoglycans – Sugar > protein Mucins (mucus) Lectins (cell-cell Three genes encode three interactions) glycosyltransferases (A B O) Glycolipid Inherited from each parent (OO, OA, OB, AB) 11  Uptake of Dietary Carbs Covers Learning Objectives: 2. Describe the digestion and absorption of the major carbohydrates, including disaccharides and polysaccharides 3. Contrast the roles of the different glucose transporters, their location and identify which are insulin regulated Digestion of Dietary Carbohydrates CHO-fig3 (1) (2) (3) α-Amylase (4) (5) (6) 13 Fig. 21.2, Marks’ 5th Ed.  Brush-Border Glycosidases (FYI) COMPLEX CATALYTIC SITES PRINCIPAL ACTIVITIES Split α-1,4-glycosidic bonds between glucosyl units, beginning sequentially with the residue at the tail β-Glucoamylase α-Glucosidase end (nonreducing end) of the chain. This is an exoglycosidase. Substrates include amylose, amylopectin, glycogen, and maltose. Same as above but with slightly different β-Glucosidase specificities and affinities for the substrates Sucrase Sucrase–maltase Splits sucrose, maltose, and maltotriose Splits α-1,-6-bonds in several limit dextrins as well as Isomaltase Isomaltase–maltase the α-1,4-bonds in maltose and maltotriose Splits β-glycosidic bonds between glucose or galactose and hydrophobic residues, such as the β-Glycosidase Glucosyl–ceramidase glycolipids glucosylceramide and galactosylceramide; also known as phlorizin hydrolase for its activity on an artificial substrate Splits the β-1,4-bond between glucose and Lactase galactose; to a lesser extent also splits the β-1,4- bond between some cellulose disaccharides Splits bond in trehalose, which is two glucosyl units Trehalase Trehalase linked α-1,1 through their anomeric carbons  Intestinal Absorptions of Dietary Carbohydrates = Facilitated fructose transporter, GLUT 5 = Facilitated glucose transporter, GLUT 1 = Na+ - glucose co-transporter, SGLT1 = Na+, K+ - ATPase 15 Fig. 21.7, Marks’ 5th Ed.  Facilitated Transported Name of Tissue transporter Process KM distribution GLUT-1 Glucose facilitated 1 mM Constitutive transport Most tissues, brain GLUT-2 Glucose facilitated ~20 mM Liver, kidney, transport (high) pancreatic  cells GLUT-3 Glucose facilitated ~1 mM (Low) Brain, placenta, transport fetal muscle GLUT-4 Glucose facilitated 5 mM Skeletal/ Heart transport, insulin Insulin  Vmax muscle, dependent Adipocytes GLUT-5 Fructose facilitated 5 mM Small intestine, liver transport (fructose) 16  Pancreatic Glucokinase & GLUT2 Glucose uptake by GLUT 2 promotes insulin secretion 17 Figs. 23-26 & 23-27, Lehninger, 6th Ed.  GLUT4 and Insulin Glucose uptake in skeletal muscles, cardiac muscles, diaphragm and adipose tissues cells is regulated by insulin-stimulated Start here exocytosis of membranous vesicles containing GLUT4 The process is reversed by endocytosis 18 Fig. 22.8 16.23 (Voet, Voet & Pratt, 3rd Ed.)  Glycolysis Covers Learning Objectives: 4. List the 3 steps of glycolysis that are regulated, where they are located and explain how they are regulated 5. Compare and contrast the roles and location of hexokinase and glucokinase 6. Explain the overall stoichiometry of glycolysis 8. Clearly contrast substrate-level phosphorylation with oxidative phosphorylation  Glycolysis Often broken into 2 stages: preparatory phase & payoff phase Some tissues (brain, kidney medulla & rapidly contracting skeletal muscles) and some cells (erythrocytes & sperm cells) rely on glucose for energy Pyruvate product can be used in subsequent pathways to produce more energy All enzymes are located in the cytosol 20 Fig. 14.2, Lehninger, 6th Ed.  Glycolysis – Stage 1 Enzymes 1. Hexokinase & Glucokinase 2. Phosphohexose isomerase 3. Phosphofructokinase-1 (PFK-1) 4. Fructose bisphosphate aldolase 5. Triose phosphate isomerase Insulin facilitates uptake of glucose in skeletal muscles, cardiac muscles, diaphragm and adipose tissues 21 Fig 15.7 (Voet, Voet & Pratt, 3rd Ed.) Glycolysis – Stage 2 Enzymes 6. Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) 7. Phosphoglycerate kinase 8. Phosphoglycerate mutase 9. Enolase 10.Pyruvate kinase Step 6 generates 2 NADH molecules Steps 7 and 10 generate 4 ATP molecules total 2 Pyruvate molecules generated 22 Fig. 15.15 (Voet, Voet & Pratt, 3rd Ed.)  Net Reaction of Glycolysis Net of 2 molecules of ATP generated (4 were made, but 2 used earlier) 2 molecules of NAD+ are reduced to NADH Glucose + 2 ADP + 2 NAD+ + 2 Pi → 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O No O2 utilized, so an anaerobic pathway 23  Regulation of Glycolysis Hexokinase/ Glucokinase Phosphofructokinase-1 (PFK-1) Pyruvate kinase 24 Glucose Transport Across Membrane 25  Hexokinase vs Glucokinase Hexokinase Hexokinase Glucokinase (Liver and pancreatic β-cells) (universal) (Most tissue) Glucose phosphorylation catalyzed by different kinases Hexokinase - exists in most tissues and inhibited by G6P Glucokinase - liver/pancreatic β-cell specific, inhibited by F6P 26 Hexokinase (Km=0.05 mM), Glucokinase (Km=10 mM), constitutive inducible Inhibited by glucose-6-phosphate An isoenzyme of hexokinase High concentrations of G-6-P signal Inducible in liver and pancreas, that the cell no longer requires provides G6P for the synthesis of glucose for energy; the glucose will be glycogen or as a source of 27 left in the blood biosynthetic precursors  Hepatic Glucokinase Regulation Regulator Protein (Glucokinase RP, aka GKRP) 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 When 𝐹6𝑃 inside hepatocyte decreases, GKRP binds glucokinase and takes it into the nucleus (inactivates its function) In high [glucose] inside hepatocyte, glucokinase released to cytoplasm 28 Fig. 15.15, Lehninger, 6th Ed.  PFK-1 Regulation PFK-1 is the rate-limiting enzyme in all tissues Allosteric activators: AMP & fructose-2, 6- bisphosphate (F-2,6-BP) W/O F-2,6-BP or AMP Allosteric inhibitors: citrate & ATP 29  F-2,6-BP (fructose-2,6-bisphosphate) Glucose PFK-2 Fructose-6-P Fructose-2,6-bisP F-2,6-bisPase + PFK-1 Fructose-1,6-bisP Pyruvate F-2,6-BP enhances PFK-1 activity F-2,6-BP levels are determined by PFK-2 and F-2,6-BPase 30  PFK-2 & F-2,6-BPase PFK-2 and F2,6-BPase can be phosphorylated and dephosphorylated Heart and skeletal muscle isozymes regulated by [substrate] PFK-2 - Liver Phosphorylated - inactive Dephosphorylated - active F2,6-Bpase - Liver Phosphorylated - active Fructose-2,6-BP Dephosphorylated - inactive Liver - regulates glycolysis and gluconeogenesis Adipose tissue - regulates glycolysis 31  Phosphofructokinase-1 - – Regulated allosterically by AMP & F-2,6-BP Skeletal muscle PFK-2 – (no phosphorylation site) – Regulated by substrate availability Liver PFK-2 – cAMP - PKA phosphorylation site 32  Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) Reversible step (also present in gluconeogenesis) Produces the high-energy 1,3-BPG and reduces NAD+ to NADH Inactivated by reaction with iodoacetate, iodoacetamide, arsenite or Hg2+ AsO43- substitutes for PO43-, creates futile cycle (potential treatment for cancer cells that rely heavily on glycolysis) p. 551 , Lehninger, 6th Ed.  Phosphoglycerate Kinase (PGK) Reversible step (also present in gluconeogenesis) Mg2+ dependent – most reactions involving ATP are Substrate level phosphorylation – ATP is made at the substrate level, not utilizing the electron transport chain (not dependent on O2) 34 p. 552 , Lehninger, 6th Ed.  Pyruvate Kinase Regulation Allosteric inhibitor – Alanine & ATP Allosteric activator – F-1,6-BP (feed-forward activation) Substrate level phosphorylation to make ATP Spontaneous conversion of “enol” pyruvate into “keto” pyruvate Branch point  Most Activator F1,6BP Inhibitors 2 ATP PP: protein Acetyl-CoA phosphatase Long-chain (insulin) FAs Alanine Liver PK is inactive when phosphorylated cAMP-dependent PKA activated by glucagon M1 isozyme in brain, heart and muscle has no allosteric sites (not major sites of gluconeogenesis) 36 Fig. 15-21, Lehninger, 6th Ed. Summary of Glycolysis Regulation 37  Lactate and Metabolism of Other Sugars Covers Learning Objectives: 7. Contrast the fate of pyruvate under aerobic vs. anaerobic metabolism and explain why NAD+ must be recycled and how 9. Describe how sugars (galactose, lactose, fructose) other than glucose are metabolized  Anaerobic Fate of Pyruvate Muscles and RBCs of vertebrates convert pyruvate into lactate and NAD+ (LDH) Under anaerobic glycolysis, the overall set of reactions can be written: Glucose + 2 ADP + 2 Pi → 2 lactate + 2 ATP + 2 H2O + 2 H+ Regenerates NAD+ for use by GAPDH 39 p. 563 & 23.19, Lehninger, 6th Ed.  Cori Cycle Lactate from muscles and RBCs is transported to the liver Liver lactate dehydrogenase (LDH) reconverts lactate to pyruvate NAD+/NADH ratio determines the direction Lactic acidosis can result from insufficient O2 Increase in lactic acid and decrease in blood pH 40  Warburg Effect Increased lactate production in cancer cells even under aerobic conditions – Increased glycolysis – More intermediary molecules for anabolism and cell growth – Acidity helps cancer cell invasion and escaping from the immune system Metabolism of Other Dietary Carbohydrates PFK-1 42 Normal Lactose/Galactose Metabolism 3 main enzymes Galactokinase (GALK) Galactose → Gal-1-P Gal 1-P uridyl transferase (GALT) Gal-1-P + UDP-Glc → UDP-Gal + G1P UDP-Gal epimerase (GALE) UDP-Gal → UDP-Glucose UPD-galactose can be used for energy or other synthetic pathways (i.e. glycoproteins, glycolipids, glycosaminoglycans) Image taken from Kaplan COMLEX-1 study book Lactose & Galactose Food Sources 44  Fructose Metabolism Fructose bypasses rate-limiting step of glycolysis (PFK-1), less regulated Consumption of high-fructose corn syrup (~50% fructose) Fatty liver Hyperglycemia Fructokinase deficiency → Fructosuria Aldolase B deficiency → Hereditary fructose intolerance 45 Fructokinase deficiency Fructosuria Rare but benign condition (autosomal recessive) Affected individuals have high blood [fructose] Aldolase B (fructose-1 phosphate aldolase) deficiency Hereditary fructose intolerance (autosomal recessive) Rate limiting step of fructose catabolism Accumulate fructose 1-P in their livers Depletes liver phosphate levels (trapped as F1P) Symptoms are severe Diarrhea, vomiting, failure to thrive Liver and kidney damage If left untreated could lead to death Dietary restrictions – avoid fruits, fruit juices, maple syrup, etc. 46 Fructose Food Sources 47 PPP, PDH Complex, & TCA Cycle Dr. Sreenilayam Oct. 2, 2024 Reading: Marks’ Basic Medical Biochemistry, 5th Ed. Ch. 23, 27 Some slides modified from Dianzheng Zhang, PhD and Francis Jenney, PhD This is the material you are responsible for: CBFM Lectures from Sept. 19 - Present 2  PPP, PDH Complex & TCA Cycle: Learning Objectives o By the end of this unit, the successful student will be able to do the following, as measured by multiple choice examination: 1. Describe the composition (including the 5 cofactors), role, mechanism and regulation of the PDH complex 2. Explain the symptoms and treatments for PDH deficiency 3. Explain how the TCA cycle is regulated at 3 steps and its location in cells 4. Identify where energy gets invested, where energy is produced and in what form (ATP, NADH) for the TCA cycle 5. Explain catapleurotic/anapleurotic reactions and describe how they are linked to pyruvate carboxylase deficiency 6. Describe the 2 different phases of the pentose phosphate pathway and the major products, and explain how the pathway is tuned to respond to different metabolic requirements 7. Explain the etiology of G6PD deficiency 3  PDH Complex Covers Learning Objectives: 1. Describe the composition (including the 5 cofactors), role, mechanism and regulation of the PDH complex 2. Explain the symptoms and treatments for PDH deficiency  Fates of Pyruvate Non-Essential Amino Acids Fatty Acids Cholesterol TCA Cycle 5  Glucose Pentose Phosphate Pathway Glycogen Pentoses Gluconeogenesis Glycolysis Cytosol Pyruvate Pyruvate Dehydrogenase Mitochondria Acetyl-CoA Oxaloacetate Citrate TCA -ketoglutarate ATP α-ketogluatrate Dehydrogenase Succinyl-CoA 6  Transport of Pyruvate Fig. 24.15, Marks’, 5th Ed. Pyruvate is located in cytosol (glycolysis) PDH (pyruvate dehydrogenase) Complex is located in the mitochondria A channel initially moves pyruvate through the outer membrane Transporter used to move pyruvate across the inner membrane 7 http://www-3.unipv.it/webbio/anatcomp/freitas/2012-2013/16_Mitocondri_studenti.pdf  PDC (PDH Complex) Overall Reaction PDH reaction is a oxidative decarboxylation (generating both reducing equivalents and CO2) 5 cofactors (think of Vitamins lecture) participate in the complex Intermediates stay attached to the enzyme as the substrate is converted into product (substrate channeling) Irreversible reaction; no counter enzyme in mammals (reason why fatty acids can’t enter gluconeogenesis) 8 Fig. 16-2, Lehninger, 6th Ed. 5 Cofactors (The Lovely Co-enzymes For Nerds) 1. Thiamine pyrophosphate (B1) 2. Lipoic acid 3. CoA (B5, pantothenic acid) 4. FAD (B2, riboflavin) 5. NAD+ (B3, niacin) 9 PDH Catalyzed Reactions 10 Fig. 19.4, Garrett & Grisham, 4th Ed.  PDH Complex Regulation Substrate activation and Product inhibition Activators: pyruvate, CoA and NAD+ Inhibitors: acetyl-CoA and NADH 11 PDH Complex Regulation Activator - insulin (liver) Covalent modification via hormones (phosphorylation and dephosphorylation) 12 PDH Complex Regulation Allosteric regulation 13  PDH Deficiency Symptoms: Increased levels of pyruvate, lactate, alanine Chronic lactic acidosis Severe neurological defects which could eventually result in death Remedies: Diet with reduced carbohydrates Dichloroacetate, an inhibitor of the PDH kinase Dichloroacetate Pyruvate 14  TCA/Krebs Cycle Covers Learning Objectives: 3. Explain how the TCA cycle is regulated at 3 steps and its location in cells 4. Identify where energy gets invested, where energy is produced and in what form (ATP, NADH) for the TCA cycle 5. Explain catapleurotic/anapleurotic reactions and describe how they are linked to pyruvate carboxylase deficiency  TCA & ETC Overview Fig. 22.10, Marks’, 5th Ed. TCA cycle is in the mitochondrial matrix (except succinate dehydrogenase in the inner membrane) ETC & ATP Synthase in the inner membrane Electrochemical gradient drives the formation of ATP  Citric Acid Cycle Reactions 8 reactions result in the reduction of NAD+ and FAD which transfer their electrons to oxygen during oxidative phosphorylation Add 2-C acetate to 4-C oxaloacetate to make 6-C citrate Oxidize and decarboxylate, generating 2 CO2 (waste), 3 NADH, and FADH2, and a GTP as energy Regenerate 4C oxaloacetate, and start again Fig. 17.2, Voet, Voet & Pratt, 3rd Ed.  Citrate Synthase Combine acetyl-CoA with oxaloacetate (OAA) to form citrate Irreversible reaction Commitment step for TCA cycle Inhibitors: NADH (allosteric), ATP (allosteric), citrate (allosteric) & succinyl- CoA (product of 4th step; negative feedback; allosteric) Activator: ADP (allosteric) Recall that citrate inhibits PFK-1 activity of glycolysis Fig. 16-9, Lehninger, 6th Ed.  Isocitrate Dehydrogenase (IDH) Fig. 16.11, Lehninger, 5th Ed. Convert isocitrate to -ketoglutarate and NAD+ is reduced to NADH Irreversible Rate limiting step Produces the first CO2 and NADH of the TCA cycle 3 isozymes IDH1 & IDH2 – NADP+-dependent (reversible, no allosteric modifiers) IDH3 - NAD+-dependent and only in mitochondrial matrix IDH3 inhibitors: NADH (allosteric) & ATP (allosteric) IDH3 activators: ADP (allosteric) & Ca2+ (allosteric)  -Ketoglutarate Dehydrogenase Converts -ketoglutarate to succinyl-CoA and yields NADH & CO2 Irreversible Produces the second NADH and CO2 molecules Resembles the PDH complex reaction E3 (dihydrolipoyl dehydrogenase) is identical Inhibitors: NADH & succinyl-CoA (both are product inhibition) Activator: Ca2+ (allosteric) Page 644 Lehninger, 6th Ed.  + Ca2+ Regulation Know which 5 enzymes (3 TCA , PDH complex, + ADP PC) are regulated and how! Know where energy and key metabolites go in and out of the cycle + Ca2+ It is mostly regulated by the ratios of: ATP : ADP (or AMP) + Ca2+ NADH : NAD+ Fig. 19.18, Garrett & Grisham, 4th Ed.  Anaplerotic & Cataplerotic Reactions Anaplerosis: (anaplerotic reaction) Synthesizing and replenishing the intermediates of the TCA cycle Cataplerosis: (cataplerotic reaction) Intermediates escaping from the TCA cycle and synthesizing other molecules 22 Fig. 19.16, Garrett & Grisham, 4th Ed. From the previous slide, know the following: Acetyl-CoA → fatty acids -ketoglutarate → Glutamate Succinyl-CoA → -aminolevulinate Fumarate/oxaloacetate → Aspartate  PPP/HMP Shunt Pentose phosphate pathway (PPP) Hexose monophosphate shunt (HMP Shunt) Phosphogluconate pathway Covers Learning Objectives: 6. Describe the 2 different phases of the pentose phosphate pathway and the major products, and explain how the pathway is tuned to respond to different metabolic requirements 7. Explain the etiology of G6PD deficiency  PPP Overview (irreversible) Occurs in the cytoplasm [NADPH] extremely critical!! Used for: (reversible) Synthesis of molecules Production of fat (storage of electrons) Protection against oxidative stress (ROS) Detoxification of xenobiotics 25 Fig. 27.1, Marks’ 5th Ed. G6P Dehydrogenase Glucose-6-P Dehydrogenase* Irreversible 1st step Produces NADPH Highly regulated *G6PD deficiency – most common genetic disease in the world! 26  PPP – Non-Oxidative Phase Basically shuffling carbons using Epimerases, Isomerases, Transketolases, and transaldolases to make many different sugars (most important Ribose-5-P) (Vit B1) 27 Fig. 27.6, Marks’ 5th Ed.  Regulation of PPP/HMP Shunt NADPH: an inhibitor of Glucose-6- P Dehydrogenase (G6PD) Liver G6PD is an inducible enzyme  insulin/glucagon (after a high carbohydrate meal) favors the PPP The PPP loops back to glycolysis pathway 28  PPP – Modified as Needed Pathway flow can be tuned to different needs, not just NADPH production (only steps 1 & 3 are irreversible) Need NADPH but no R5P Need R5P but no NADPH Need R5P and NADPH Need ATP and NADPH but no R-5-P Need some other sugar(s) 29 Fig. 15.35 Voet, Voet & Pratt, 3rd Ed.  G6PD Deficiency An X-linked recessive disease Most common human enzyme defect Usually occurs in males More common in African and Mediterranean decent Hemolysis induced by oxidative stresses Triggers: infections, stress, fava beans, aspirin, and other drugs Symptoms: fever, dark urine, abdominal and back pain, fatigue, and pale skin 30 ETC & Oxidative Phosphorylation Dr. Sreenilayam Oct. 3, 2024 Reading: Marks’ Basic Medical Biochemistry, 5th Ed. Ch. 20, 24 Some slides modified from Kimberly Baker, PhD and Dianzheng Zhang, PhD This is the material you are responsible for: Cell Membrane Components, and Transport Across Membranes (CBFM) Histology of the cell (CBFM) CBFM Lectures from Sept. 19 - Present 2  ETC & Oxidative Phosphorylation: Learning Objectives o By the end of this unit, the successful student will be able to do the following, as measured by multiple choice examination: 1. Explain the overall structure and function of the ETC and describe what its ‘circuits’ (Fe-S clusters, flavins, cytochromes, etc) are made of 2. Describe the Q-cycle, why and how it occurs 3. Contrast between substrate-level and oxidative phosphorylation for ATP production 4. Identify the nutritional requirements important for the ETC 5. Illustrate precisely where oxygen is needed in the ETC and other roles of oxygen in the body 6. Identify the ETC protein the following poisons act on: arsenic, cyanide, aspirin, 2,4 - DNP, oligomycin, sodium thiosulfate, CO, azide, doxorubicin, rotenone, antimycin A 7. Explain uncoupled oxidative phosphorylation and its importance 8. Explain the purpose of brown fat, describe how and why is it ‘brown’, and compare and contrast to ‘uncouplers’ 9. Explain the regulation of oxidative phosphorylation 10. Describe how ADP and ATP are transported across the mitochondrial inner membrane 11. Describe how cytosolic reducing equivalents of NADH are shuttled across the 3 mitochondrial membrane  Transport Across the Mitochondrial Membranes Covers Learning Objectives: 10. Describe how ADP and ATP are transported across the mitochondrial inner membrane 11. Describe how cytosolic reducing equivalents of NADH are shuttled across the mitochondrial membrane  Mitochondria Powerhouse of the cell Generates ATP Number of mitochondria in cells varies Ranges from 100’s to 1,000’s Can take up to 20% of the cell Muscles have most Adipose have some RBCs have none 5  Mitochondrial Structure mitos = thread, chondros = granule Fig. 18.2, Voet, Voet & Pratt, 3rd Ed. Outer membrane is permeable and contains unspecific pores - Transmembrane protein that permits passive diffusion of molecules MW