1 / 57

CHAPTER 6 Enzymes

CHAPTER 6 Enzymes. 6.1 An Introduction to Enzymes Catalysts, Proteins, Reaction Types 6.2 How Enzymes Work Rate Enhancement, Activation energy, Catalytic Mechanism 6.3 Enzyme Kinetics Steady-State, Michaelis-Menten Formalism Inhibition 6.4 Examples of Enzymatic Reactions

bernard
Télécharger la présentation

CHAPTER 6 Enzymes

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. CHAPTER 6Enzymes 6.1 An Introduction to Enzymes • Catalysts, Proteins, Reaction Types 6.2 How Enzymes Work • Rate Enhancement, Activation energy, Catalytic Mechanism 6.3 Enzyme Kinetics • Steady-State, Michaelis-Menten Formalism • Inhibition 6.4 Examples of Enzymatic Reactions 6.5 Regulatory Enzymes

  2. 6.1 What are Enzymes? Enzymes are catalytically active biological macromolecules Specific, Efficient, Active in Aqueous Solution Most enzymes are globular proteins, however some RNA (ribozymes, and ribosomal RNA) also catalyze reactions Non-peptide Co-Factors (Metals, Vitamins, Coenzymes) Enzymes can be classified functionally

  3. Carbonic Anhydrase Tissues Lungs and Kidney 107 rate enhancement

  4. Mammalian Carbonic anhydrases- 15 genes, widely distributed • Comparison of mammalian carbonic anhydrases • Isoform, Gene MW[6] Location (cell)Location (tissue)[6] Specific activity • CA-I CA1 29 kDa cytosol red blood cell and GI tract 2.0 × 10e5 • CA-II CA2 29 kDa cytosol almost ubiquitous 1.4 × 10e6 • CA-III CA3 29 kDa cytosol 8% of soluble protein muscle 1.3 × 10e4 • CA-IV CA4 35 kDa extr GPI GI tract, kidney, endothelium 1.1 × 10e6 • CA-VACA5A 35kDa (pr) mitochondria liver 2.9 × 10e5 • CA-VBCA5B 36 kDa (pr) mitochondria widely distributed 9.5 × 10e5 • CA-VI CA6 39-42 kDa secretory saliva and milk 3.4 × 10e5 • CA-VII, CA7 29 kDa cytosol widely distributed 9.5 × 10e5 • CA-IX CA9 54, 58 kDa mem-assoc normal GI tract, cancers 1.1 × 10e6 • CA-XII, CA12 44 kDa extra-(active site), kidney, cancers 4.2 × 10e5 • CA-XIII , CA13 29 kDa cytosol widely distributed 1.5 × 10e5 • CA-XIV, CA14 54 kDa eextra-(active site), kidney, heart, muscle, brain 3.1 × 10e5 • CA-XV , CA15 34-36 kDa GPI-linked kidney, not expressed in human tissues4.7 × 10e5

  5. Why Biocatalysis? • Higher reaction rates • Greater reaction specificity • Milder reaction conditions • Capacity for regulation • Metabolites have many potential pathways of decomposition • Enzymes make the desired one most favorable

  6. Enzymatic Substrate Selectivity No binding Binding but no reaction Example: Phenylalanine hydroxylase

  7. Enzymes affect rates but not equilibria Kinetics and Equilibrium

  8. Transition State Theory • Slow reactions face significant activation barriers that must be surmounted during the reaction • transition state theory is applicable for catalysis • rate constants and free energies can be related

  9. Rate Acceleration • The enzyme lowers the activation barrier compared to the uncatalyzed aqueous reaction

  10. How to Lower G‡? Enzymes organizes reactive groups into proximity • Uncatalyzed bimolecular reactions: • two free reactants  single restricted transition state • conversion is entropically unfavorable • Uncatalyzed unimolecular reactions: • flexible reactant  rigid transition state conversion is • entropically unfavorable for flexible reactants • Catalyzed reactions: • Enzyme uses the binding energy of substrates to organize • the reactants to a fairly rigid ES complex • Entropy cost is paid during binding • Rigid reactant complex  transition state conversion is entropically OK

  11. Support for the Proximity Model • The rate of anhydride formation from esters and carboxylates shows a strong dependence on proximity of two reactive groups (work by Thomas C. Bruice’s group)

  12. Support for TS Stabilization • Stable structural analogs of transition states bind more strongly than reactants • Antibodies to TS analogs can have catalytic activity

  13. TS Stabilization Cartoon

  14. How is TS Stabilization Achieved? • acid-base catalysis: give and take protons • covalent catalysis: change reaction paths • metal ion catalysis: use redox cofactors, pKa shifters • electrostatic catalysis: preferential interactions with TS

  15. Acid-base Catalysis: Chemical Example Consider ester hydrolysis: Water is a poor nucleophile, and methanol is a poor leaving group Aqueous hydrolysis can be catalyzed either by acids or by bases Enzymes can do acid and base catalysis simultaneously

  16. General Acid-Base Catalysis • Example: amide hydrolysis

  17. Amino Acids in General Acid-Base catalysis

  18. Covalent Catalysis: Chemical Example • The anhydride hydrolysis reaction is catalyzed by pyridine, a better nucleophile than water (pKa=5.5). • Hydrolysis is accelerated because of charge loss in the transition state makes pyridine a good leaving group.

  19. Covalent Catalysis: In Enzymes • Proteases and peptidases • chymotrypsin, elastase, subtilisin • reactive serine nucleophile • Some aldehyde dehydrogenase • glyceraldehyde-3phosphate dehydrogenase • reactive thiolate nucleophile • Aldolases and decarboxylases • amine nucleophile • Dehalogenases • carboxylate nucleophile

  20. Enzymes affect rates but not equilibria Reactions are Complex – Simplifying assumptions Enzyme Kinetics

  21. Pre-Equilibrium and Steady State Pre- EQ: assoc. & dissoc. Faster than catalysis Steady State: ES intermediate, [P]=0

  22. Michaelis-Menten kinetics • Total Enzyme concentration is fixed • ET = [E] + [ES]

  23. Km => Kd when k-1 >> k2 Michaelis Constant Km

  24. Enzyme-Substrate Complex • Enzymes act by binding substrates • the non-covalent enzyme substrate complex is known as the Michaelis complex • allows thinking in terms of chemical interactions • allows development of kinetic equations

  25. Initial Rates, v0 Linear region [S]≅[S]0 [P] ≅ 0 Enzyme kinetics saturable V0 = Vmax when [S]= ∞

  26. Why Study Enzyme Kinetics? • Quantitative description of biocatalysis • Determine the order of binding of substrates • Elucidate acid-base catalysis • Understand catalytic mechanism • Find effective inhibitors • Understand regulation of activity

  27. Effect of Substrate Concentration • Ideal Rate: • Deviations due to: • Limitation of measurements • Substrate inhibition • Substrate prep contains inhibitors • Enzyme prep contains inhibitors

  28. Determination of Kinetic Parameters Linearized double-reciprocal plot is good for analysis of two-substrate data or inhibition

  29. Identify Constraints and Assumptions • Total enzyme concentration is constant • Mass balance equation for enzyme: ETot = [E] + [ES] • It is also implicitly assumed that: STot = [S] + [ES] ≈ [S] • Steady state assumption: • What is the observed rate • Rate of product formation:

  30. kcat and Km • The final form in case of a single substrate is • kcat (turnover number): how many substrate molecules can one enzyme molecule convert per second • Km (Michaelis constant): an approximate measure of substrate’s affinity for enzyme • Microscopic meaning of Km and kcat depends on the details of the mechanism

  31. Two-substrate Reactions • Kinetic mechanism: the order of binding of substrates and release of products • When two or more reactants are involved, enzyme kinetics allows to distinguish between different kinetic mechanisms • Sequential mechanism • Ping-Pong mechanism

  32. Sequential Kinetic Mechanism We cannot easily distinguish random from ordered Lines intersect

  33. Ping-Pong Kinetic Mechanism Lines are parallel

  34. Enzyme Inhibition • Inhibitors are compounds that decrease enzyme’s activity • Irreversible inhibitors (inactivators) react with the enzyme • one inhibitor molecule can permanently shut off one enzyme molecule • they are often powerful toxins but also may be used as drugs • Reversible inhibitors bind to, and can dissociate from the enzyme • - they are often structural analogs of substrates or products • - they are often used as drugs to slow down a specific enzyme • Reversible inhibitor can bind: • To the free enzyme and prevent the binding of the substrate • To the enzyme-substrate complex and prevent the reaction

  35. Classification of Reversible Inhibitors • Reversible inhibitor can bind: • To the free enzyme and prevent the binding of the substrate • To the enzyme-substrate complex and prevent the reaction

  36. Competitive Inhibition Lines intersect at the y-axis

  37. Uncompetitive Inhibition Lines are parallel

  38. Mixed Inhibition Lines intersect left from the y-axis

  39. pH Profiles Reveal Active site pKa’s

  40. Chymotrypsin Mechanism Step 1: Substrate Binding Endo-peptidase specificity P3 - P2 - P1-|-P1’- P2’- P3’

  41. Chymotrypsin MechanismStep 2: Nucleophilic Attack Serine protease – catalytic triad activates serine hydroxyl

  42. Chymotrypsin MechanismStep 3: Substrate Cleavage N-terminal peptide released Covalent acyl intermediate

  43. Chymotrypsin MechanismStep 4:Water attack

  44. Chymotrypsin MechanismStep 5: De-acylation

  45. Chymotrypsin MechanismStep 6: Serine hydroxyl restored

  46. Chymotrypsin MechanismStep 7: Product Dissociates

  47. Peptidoglycan and Lysozyme • Peptidoglycan is a polysaccharide found in many bacterial cell walls • Cleavage of the cell wall leads to the lysis of bacteria • Lysozyme is an antibacterial enzyme

  48. General Acid-Base + Covalent Catalysis: Cleavage of Peptidoglycan by Lysozyme X-ray structures of lysozyme with bound substrate analogs show that the C-1 carbon is located between Glu 35 and Asp 52 residues.

More Related