Enzymes chp-6-7-bioc-361-version-oct-2012b


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  • FIGURE 6-18d Structure of chymotrypsin. (PDB ID 7GCH) (d) A close-up of the active site with a substrate (mostly green) bound. Two of the active-site residues, Ser 195 and His 57 (both red), are partly visible. The hydroxyl of Ser 195 attacks the carbonyl group of the substrate (the oxygen is purple); the developing negative charge on the oxygen is stabilized by the oxyanion hole (amide nitrogens, including one from Ser 195 , in orange), as explained in Figure 6-21. In the substrate, the aromatic amino acid side chain and the amide nitrogen of the peptide bond to be cleaved (protruding toward the viewer and projecting the path of the rest of the substrate polypeptide chain) are in blue.
  • FIGURE 6-10 Initial velocities of enzyme-catalyzed reactions. A theoretical enzyme catalyzes the reaction S ↔ P, and is present at a concentration sufficient to catalyze the reaction at a maximum velocity, V max , of 1 μ M/min. The Michaelis constant, K m (explained in the text), is 0.5 μ M. Progress curves are shown for substrate concentrations below, at, and above the K m . The rate of an enzyme-catalyzed reaction declines as substrate is converted to product. A tangent to each curve taken at time = 0 defines the initial velocity, V 0 , of each reaction.
  • FIGURE 6-31 Subunit interactions in an allosteric enzyme, and interactions with inhibitors and activators. In many allosteric enzymes the substrate binding site and the modulator binding site(s) are on different subunits, the catalytic (C) and regulatory (R) subunits, respectively. Binding of the positive (stimulatory) modulator (M) to its specific site on the regulatory subunit is communicated to the catalytic subunit through a conformational change. This change renders the catalytic subunit active and capable of binding the substrate (S) with higher affinity. On dissociation of the modulator from the regulatory subunit, the enzyme reverts to its inactive or less active form.
  • Enzymes chp-6-7-bioc-361-version-oct-2012b

    1. 1. Chapter SixThe Behavior of Proteins: Enzymes Jody Haddow - UAEU
    2. 2. 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. 3. Carbonic Anhydrase Tissues   Lungs and Kidney 107 rate enhancement
    4. 4. Why Biocatalysis?• Higher reaction rates• Greater reaction specificity• Milder reaction conditions• Capacity for regulation - -COO COO NH2 - - O COO • Metabolites haveOH COO many potential - pathways of - O COO Chorismate COO - decompositionCOO OH mutase - OOC O • Enzymes make the desired one most OHNH2 favorable
    5. 5. Enzymatic Substrate Selectivity OH H H - + OOC NH3 - + OOC NH3 H - OOC + NH3 No binding OH HO OH H H Binding but no H NH CH3 reactionExample: Phenylalanine hydroxylase
    6. 6. Enzyme Catalysis• Enzyme: a biological catalyst • with the exception of some RNAs that catalyze their own splicing (Section 10.4), all enzymes are proteins • enzymes can increase the rate of a reaction by a factor of up to 1020 over an uncatalyzed reaction • some enzymes are so specific that they catalyze the reaction of only one stereoisomer; others catalyze a family of similar reactions• The rate of a reaction depends on its activation energy, ∆G°‡ • an enzyme provides an alternative pathway with a lower activation energy
    7. 7. Enzyme Catalysis (Cont’d)• Consider the reaction H2 O 2 H2 O + O2
    8. 8. Temperature dependence of catalysis• Temperature can also catalyze reaction (increase rate)• This is dangerous, why?• Increasing temperature will eventually lead to protein denaturation
    9. 9. Michaelis-Menten Kinetics• Initial rate of an enzyme-catalyzed reaction versus substrate concentration
    10. 10. 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
    11. 11. Initial Rates, v0• Linear region• [S]≅[S]0• [P] ≅ 0• Enzyme kinetics saturable• V0 = Vmax when [S]= ∞
    12. 12. Michaelis-Menten Model• For an enzyme-catalyzed reaction k1 k2 E + S ES P k-1• The rates of formation and breakdown of ES are given by these equations rate of formation of ES = k1 [E][S] rate of breakdown of ES = k-1 [ES] + k 2[ES]• At the steady state k1 [E][S] = k -1[ES] + k 2[ES]
    13. 13. Michaelis-Menten Model (Cont’d)• When the steady state is reached, the concentration of free enzyme is the total less that bound in ES [E] = [E] T - [ES]• Substituting for the concentration of free enzyme and collecting all rate constants in one term gives ([E]T - [ES]) [S] k-1 + k2 = = KM [ES] k1• Where KM is called the Michaelis constant
    14. 14. Michaelis-Menten Model (Cont’d)• It is now possible to solve for the concentration of the enzyme-substrate complex, [ES] [E]T [S] - [ES][S] = KM [ES] [E]T [S] - [ES][S] = KM[ES] [E]T [S] = [ES](K M + [S])• Or alternatively [E] T [S] [ES] = KM + [S]
    15. 15. Michaelis-Menten Model (Cont’d) • In the initial stages, formation of product depends only on the rate of breakdown of ES k 2[E]T [S] Vinit = k2 [ES] = KM + [S] • If substrate concentration is so large that the enzyme is saturated with substrate [ES] = [E]T Vinit = Vmax = k2 [E]T • Substituting k2[E]T = Vmax into the top equation gives Vmax [S] Michaelis-Menten Vinit = KM + [S] equation
    16. 16. Michaelis-Menten Model (Cont’d) • When [S]= KM, the equation reduces to Vmax [S] Vmax [S] VmaxV= = = KM + [S] [S] + [S] 2
    17. 17. Linearizing The Michaelis-Menten Equation • It is difficult to determine Vmax experimentally • The equation for a hyperbola Vmax [S] V= (an equation for a hyperbola) KM + [S] • Can be transformed into the equation for a straight line by taking the reciprocal of each side 1 = KM + [S] KM [S] = + V Vmax [S] Vmax [S] Vmax [S] 1 = KM 1 + V Vmax [S] Vmax
    18. 18. Lineweaver-Burk Plot • The Lineweaver-Burke plot has the form y = mx + b, and is the formula for a straight line 1 KM 1 1 = • + V Vmax [S] Vmax y = m • x + b • a plot of 1/V versus 1/[S] will give a straight line with slope of KM/Vmax and y intercept of 1/Vmax • such a plot is known as a Lineweaver-Burk double reciprocal plot
    19. 19. Lineweaver-Burk Plot (Cont’d) • KM is the dissociation constant for ES; the greater the value of KM, the less tightly S is bound to E • Vmax is the turnover number
    20. 20. Turnover Numbers • Vmax is related to the turnover number of enzyme:also called kcat Vax =t r oe n me m  un v r_ u br=kt ca  E] [ T • Number of moles of substrate that react to form product per mole of enzyme per unit of time
    21. 21. Chapter Seven The Behavior of Proteins:Enzymes, Mechanisms, and Control
    22. 22. Enzymes fall into classes based on function• There are 6 major classes of enzymes: 1.Oxidoreductases which are involved in oxidation, reduction, and electron or proton transfer reactions; 2.Transferases, catalysing reactions in which groups are transferred; 3.Hydrolases which cleave various covalent bonds by hydrolysis; 4 4.Lyases catalyse reactions forming or breaking double bonds; 5.Isomerases catalyse isomerisation reactions; 6.Ligases join substituents together covalently.
    23. 23. Allosteric Enzymes• Allosteric: Greek allo + steric, other shape• Allosteric enzyme: an oligomer whose biological activity is affected by other substances binding to it • these substances change the enzyme’s activity by altering the conformation(s) of its 4°structure• Allosteric effector: a substance that modifies the behavior of an allosteric enzyme; may be an • allosteric inhibitor • allosteric activator• Aspartate transcarbamoylase (ATCase) • feedback inhibition
    24. 24. Feedback InhibitionFormation of productinhibits its continuedproduction
    25. 25. Allosteric Regulation; ATCase
    26. 26. Enzyme InhibitionInhibitors 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
    27. 27. Types of Inhibition • Competitive Inhibition • Noncompetitive Inhibition • Irreversible Inhibition
    28. 28. Competitive Inhibition Enzyme S I In competitive inhibition, the inhibitor competes with the substrate for the same binding site
    29. 29. Competitive inhibitors• Enzymes can be inhibited competitively, when the substrate and inhibitor compete for binding to the same active site• This can determined by plotting enzyme activity with and without the inhibitor present.• Competitive Inhibition• In the presence of a competitive inhibitor, it takes a higher substrate concentration to achieve the same velocities that• were reached in its absence. So while Vmax can still be reached if sufficient substrate is available, one- half Vmax requires a higher [S] than before and thus Km is larger.
    30. 30. Competitive Inhibition - Reaction Mechanism E+S ES E+P + I In competitive inhibition, EI the inhibitor binds only to the free enzyme, not to the ES complex
    31. 31. General Michaelis-Menten Equation Vmax,app [S] v= Km,app + [S] This form of the Michaelis-Menten equation can be used to understand how each type of inhibitor affects the reaction rate curve
    32. 32. In competitive inhibition, only the apparent Km is affected (Km,app> Km),The Vmax remains unchanged by the presence of the inhibitor.
    33. 33. Competitive inhibitors alter the. apparent Km, not the Vmax - Inhibitor Vmax Reaction Rate + Inhibitor Vmax 2 Vmax,app = Vmax Km,app > Km Km Km,app [Substrate]
    34. 34. The Lineweaver-Burk plot is diagnostic forcompetitive inhibition 1 = Km,app 1 + 1 Increasing [I] v Vmax [S] Vmax Km,app 1 Slope = Vmax v 1 Vmax -1 1 Km,app [S]
    35. 35. Relating the Michaelis-Menten equation, the v vs. [S] plot,and the physical picture of competitive inhibition Inhibitor S . competes with substrate, - Inhibitor Vmax . decreasing its I Reaction Rate apparent affinity: + Inhibitor Km,app > Km Vmax 2 Km,app > K m E+S ES E+P V max,app = V max + I Km Km,app Formation of EI Formation of EI [Substrate] complex shifts reaction complex shifts reaction to the left: K m,app > K EI to the left: Km,app > Kmm
    36. 36. Noncompetitive Inhibition . I I SEnzyme S Enzyme the inhibitor does not interfere with S substrate I I binding (and S vice versa)Enzyme Enzyme
    37. 37. Non-competitive inhibitor• With noncompetitive inhibition, enzyme molecules that have been bound by the inhibitor are taken out of the game so enzyme rate (velocity) is reduced for all values of [S], including Vmax and one-half Vmax but• Km remains unchanged because the active site of those enzyme molecules that have not been inhibited is unchanged.
    38. 38. Noncompetitive Inhibition - ReactionMechanism E+S ES E+P + + In noncompetitive inhibition, the I I inhibitor binds enzyme irregardless of whether the EI + S ESI substrate is bound
    39. 39. Noncompetitive inhibitors decrease .the Vmax,app, but don’t affect the Km Vmax - Inhibitor Reaction Rate Vmax,app 1 V + Inhibitor 2 max 1 V 2 max,app Vmax,app < Vmax Km,app = Km Km [Substrate] Km,app
    40. 40. Why does Km,app = Km fornoncompetitive inhibition? E+S ES E+P + + The inhibitor binds equally well to free I I enzyme and the ES complex, so it doesn’t alter apparent affinity EI + S of the enzyme for the ESI substrate
    41. 41. The Lineweaver-Burk plot is diagnostic fornoncompetitive inhibition 1 = Km 1 1 Increasing [I] + v Vmax,app [S] Vmax,app 1 Slope = Km v Vmax,app 1 Vmax,app -1 1 Km [S]
    42. 42. Relating the Michaelis-Menten equation, the v vs. [S] plot,and the physical picture of noncompetitive inhibition I I . S Enzyme S Enzyme Inhibitor doesn’t interfere with substrate binding, Km,app = K m S I I . S Vmax - Inhibitor Enzyme Enzyme Reaction Rate Vmax,app E+S ES E+P 1 + Inhibitor + + Even at high substrate 1 V V 2 max Km,app > Km V max,app < V max I I Formation of EI levels, 2 max,app inhibitor still Vmax,app = = Km K m,app Vmax complex shifts reaction binds, Km Km,app to the left: Km,app<>[ES] Km EI + S ESI [E] t [Substrate] V max,app < V max
    43. 43. Irreversible Inhibition In irreversible Enzyme inhibition, the inhibitor binds to S the enzyme O I irreversibly through formation of a covalent bond with the enzyme , permanently inactivating the enzyme
    44. 44. Irreversible Inhibition - ReactionMechanism E+S ES E+P + In irreversible I inhibition, the inhibitor permanently inactivates the enzyme. The net effect is to remove EI enzyme from the reaction. Vmax decreases No effect on Km
    45. 45. The Michaelis-Menten plot for an irreversibleinhibitor looks like noncompetitive inhibition . Vmax - Inhibitor Reaction Rate Vmax,app 1 V + Inhibitor 2 max 1 V 2 max,app Vmax,app < Vmax Km,app = Km Km [Substrate] Km,app
    46. 46. Irreversible inhibition is distinguished fromnoncompetitive inhibition by plotting Vmax vs [E]t . tor i bi tor Enzyme is nh tor i bi inactivated hib eI Inhi - In until all of the iblVmax ble itive rsi et rs ve mp irreversible ve Re co inhibitor is rr e + n No +I used up [E]t [E]t < [I] [E]t > [I] [E]t = [I]
    47. 47. Summary-Enzyme Inhibition• Competitive Inhibitor • Binds to substrate binding site • Competes with substrate • The affinity of the substrate appears to be decreased when inhibitor is present (Km,app >Km)• Noncompetitive inhibitor • Binds to allosteric site • Does not compete with the substrate for binding to the enzyme • The maximum velocity appears to be decreased in the presence of the inhibitor (Vmax,app <Vmax)• Irreversible Inhibitor • Covalently modifies and permanently inactivates the enzyme
    48. 48. Competitive/noncompetitive inhibitor
    49. 49. Effect of inhibitors
    50. 50. Enzyme Regulation • Allosteric regulation, • heterotropic ligand binding modulates substrate binding and catalysis, • Feedback regulates metabolic pathways • Covalent modification – Reversible • Phosphorylation, nucleotides, lipid anchors • Proteolysis converts inactive pro-enzymes (zymogens) to active
    51. 51. Allosteric Enzymes• Effector molecules change the activity of an enzyme by binding at a second site • Some effectors speed up enzyme action (positive allosterism) • Some effectors slow enzyme action (negative allosterism)
    52. 52. Protein Modification• In protein modification a chemical group is covalently added to or removed from the protein • Covalent modification either activates or turns off the enzyme• The most common form of protein modification is addition or removal of a phosphate group • This group is located at the R group (with a free – OH) of: • Serine • Threonine • Tyrosine
    53. 53. Control of Enzyme Activity via Phosphorylation• The side chain -OH groups of Ser, Thr, and Tyr can form phosphate esters• Phosphorylation by ATP can convert an inactive precursor into an active enzyme• Membrane transport is a common example
    54. 54. Covalent ModificationLipase:
    55. 55. Proenzymes• A proenzyme, an enzyme made in an inactive form• It is converted to its active form • By proteolysis (hydrolysis of the enzyme) • When needed at the active site in the cell • Pepsinogen is synthesized and transported to the stomach where it is converted to pepsin
    56. 56. Coenzymes• Coenzyme: a nonprotein substance that takes part in an enzymatic reaction and is regenerated for further reaction • metal ions- can behave as coordination compounds. (Zn2+, Fe2+) • organic compounds, many of which are vitamins or are metabolically related to vitamins (Table 7.1).
    57. 57. NAD+/NADH• Nicotinamide adenine dinucleotide (NAD+) is used in many redox reactions in biology.• Contains:1) nicotinamide ring2) Adenine ring3) 2 sugar-phosphate groups
    58. 58. NAD+/NADH (Cont’d)• NAD+ is a two-electron oxidizing agent, and is reduced to NADH• Nicotinamide ring is where reduction-oxidation occurs