Enzyme Specificity and Regulation: Mechanisms and Models in Biochemical Processes

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

11. Blood Clotting Cascade

12. 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

14. 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

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

16. 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

17. Feedback Inhibition

19. 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

20. 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)

22. 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

24. Phosphoglucomutase

26. 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)

29. 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)

32. 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

35. 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

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

38. 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

42. 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

43. 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

45. 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!

46. 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)

47. O2 Binding Curves for Myoglobin and Hemoglobin