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Chapter 15

Chapter 15. Enzyme Specificity and Regulation. All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777. Outline.

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Chapter 15

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  1. Chapter 15 Enzyme Specificity and Regulation All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

  2. Outline • 15.1 Specificity from Molecular Recognition • 15.2 Controls over Enzymatic Activity • 15.3 Allosteric Regulation of Enzyme Activity • 15.4 Allosteric Model - we will only cover part of this section • Special Topic Purine Nucleoside Phosphorylase • 15.5 Glycogen Phosphorylase • SPECIAL FOCUS: Hemoglobin and Myoglobin - we will only cover part of this special topic

  3. 15.1 Specificity The Result of Molecular Recognition • Substrate (small) binds to enzyme (large) via weak forces - what are they? • H-bonds, van der Waals, ionic • Hydrophobic interactions • What are the lock-and-key and induced-fit models??? • How does induced-fit relate to transition states???

  4. Induced Fit

  5. Transition State Model • Enzyme/substrate complex is a dynamic structure • Enzyme undergoes conformational change (active conformation) upon binding of substrate(s) that causes substrate(s) to adopt a form that mimics the transition state of the reaction • Enzyme binding affinity is optimal for transition state - first proposed by Linus Pauling

  6. 15.2 Controls over Enzyme Activity Key Features: • Rate slows as product accumulates • equilibrium reached; product inhibition • Rate depends on substrate availability • Km’s of enzymes often is close to in vivo concentration of substrate • remember that when [S]=Km; v=Vmax/2 • Genetic controls - induction and repression • constitutive and inducible forms (isozymes)

  7. 15.2 Controls over Enzyme Activity (cont’d • Enzymes can be modified covalently to modulate activity - e.g., by phosporylation • Allosteric effectors can be a factor • molecules that bind to “another site” • Pro-enzymes (also known as Zymogens), isozymes and modulator proteins can also be important

  8. Blood Clotting Cascade

  9. Blood Clotting Cascade • Note that seven of the clotting factors are serine proteases -> Which means what??? • Thrombin cleaves fibrinogen so that negatively charged amino acids are lost to proteolysis to give fibrin • Fibrin readily aggregates into ordered fibrous arrays that are then cross linked to form the actual clot • Thrombin is homologous to trypsin

  10. LDH Isozymes • Muscle LDH (A4 form) works best in NAD+ generating direction, i.e., pyruvate to lactate • Heart LDH (B4 form) works best in the opposite direction -> lactate to pyruvate

  11. Modulator Proteins (for example, cyclic AMP dependent protein kinase)

  12. 15.3 Allosteric Regulation Action at "another site" • Enzymes situated at key steps in metabolic pathways are modulated by allosteric effectors • These effectors are usually produced elsewhere in the pathway • Effectors may be feed-forward activators or feedback inhibitors • Kinetics show sigmoid ("S-shaped") plots

  13. Feedback Inhibition

  14. Models for Allosteric Behavior • Monod, Wyman, Changeux Model: allosteric proteins can exist in two states: R (relaxed) and T (taut) • In this model, all the subunits of an oligomer must be in the same state • T state predominates in the absence of substrate S • S binds much tighter to R than to T

  15. Models for Allosteric Behavior • Cooperativity is achieved because S binding increases the population of R, which increases the sites available to S • Ligands such as S are positive homotropic effectors (can also have negative effectors) • Molecules that influence the binding of something other than themselves are heterotropic effectors - can be either positive (allosteric activators) or negative (allosteric inhibitors)

  16. Glycogen PhosphorylaseAllosteric Regulation and Covalent Modification • GP cleaves glucose units from nonreducing ends of glycogen • A phosphorolysis reaction is involved • Muscle GP is a dimer of identical subunits, each with PLP (pyridoxal phosphate) covalently linked • There is an allosteric effector site at the subunit interface

  17. Phosphoglucomutase

  18. Glycogen PhosphorylaseAllosteric Regulation and Covalent Modification • Pi is a positive homotropic effector • ATP is a feedback inhibitor - negative heterotropic effector (allosteric inhibitor) • Glucose-6-P is a feedback inhibitor -negative heterotropic effector(allosteric inhibitor) • AMP is a positive heterotropic effector (i.e., an allosteric activator)

  19. Regulation of GP by Covalent Modification • In 1956, Edwin Krebs and Edmond Fischer showed that a ‘converting enzyme’ could convert phosphorylase b to phosphorylase a • Three years later, Krebs and Fischer show that this conversion involves covalent phosphorylation • This phosphorylation is mediated by an enzyme cascade (Figure 15.19)

  20. cAMP is a Second Messenger • Cyclic AMP is the intracellular agent of extracellular hormones - thus a ‘second messenger’ • Hormone binding stimulates a GTP-binding protein (G protein), releasing G(GTP) • Binding of G(GTP) stimulates adenylyl cyclase to make cAMP

  21. Myoglobin and Hemoglobin • Hemoglobin and myoglobin are oxygen transport and storage proteins • Myoglobin is monomeric; hemoglobin is tetrameric • Mb: 153 aa, 17,200 MW • Hb: two alpha chains of 141 residues, 2 beta chains of 146

  22. Both Myoglobin and Hemoglobin contain hemeSee Figure 5.15 in your text

  23. Myoglobin Structure Mb is a monomeric heme protein • Mb polypeptide surrounds the heme group • Fe in Mb is Fe2+ - ferrous iron - the form that binds oxygen • Oxidation of Fe yields 3+ charge - ferriciron -metmyoglobin does not bind oxygen • Oxygen binds as the sixth ligand to Fe

  24. Hemoglobin FunctionHb must bind oxygen in lungs and release it in capillaries • When a first oxygen binds to Fe in heme of Hb, the heme Fe is drawn into the plane of the porphyrin ring • This initiates a series of conformational changes that are transmitted to adjacent subunits

  25. Hemoglobin FunctionHb must bind oxygen in lungs and release it in capillaries • Thus, the adjacent subunits' affinity for oxygen increases • This is often referred to as positive cooperativity

  26. The Conformation Change How Hemoglobin Works! • Oxygen binding changes the protein conformation • Without oxygen bound, Fe is out of heme plane • Oxygen binding pulls the Fe into the heme plane • Fe pulls its His F8 ligand along with it • The F helix moves when oxygen binds • Total movement of Fe is 0.029 nm - 0.29 Å • This change means little to Mb, but lots to Hb!

  27. Binding of Oxygen by Hb The Physiological Significance • Hb must be able to bind oxygen in the lungs • Hb must be able to release oxygen in capillaries • The sigmoid, cooperative oxygen binding curve of Hb makes this possible! • So at high O2 concentrations, hemoglobin’s affinity for O2 is high (in lungs) • At low O2 concentrations, hemoglobin’s affinity for O2 is low (in capillaries)

  28. O2 Binding Curves for Myoglobin and Hemoglobin

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