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Enzyme Inhibition and Mechanisms

Enzyme Inhibition and Mechanisms. Andy Howard Introductory Biochemistry, Fall 2010 Monday 20 September 2010. How do enzymes reduce activation energies?. We want to understand what is really happening chemically when an enzyme does its job.

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Enzyme Inhibition and Mechanisms

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  1. Enzyme Inhibition and Mechanisms Andy HowardIntroductory Biochemistry, Fall 2010Monday 20 September 2010 Biochem: Inhibition & Mechanisms

  2. How do enzymes reduce activation energies? • We want to understand what is really happening chemically when an enzyme does its job. • We’d also like to know how biochemists probe these systems. Biochem: Inhibition & Mechanisms

  3. Enzyme kinetics, concluded Inhibition Reversible & Not Categories Kinetics Drug Design Mechanisms Terminology Transition States Diffusion-controlled Reactions Binding Modes Intermediates Types of reactions Inhibition & Mechanism Topics Biochem: Inhibition & Mechanisms

  4. iClicker quiz question #1 • If we alter the kinetics of a reaction by increasing Km but leaving Vmax alone, how will the L-B plot change? Biochem: Inhibition & Mechanisms

  5. iClicker question 2 • Enzyme E has a tenfold stronger affinity for substrate A than for substrate B. Which of the following is true? • (a) Km(A) = 10 * Km(B) • (b) Km(A) = 0.1 * Km(B) • (c) Vmax(A) = 10 * Vmax(B) • (d) Vmax(A) = 0.1 * Vmax(B) • (e) None of the above. Biochem: Inhibition & Mechanisms

  6. Another physical significance of Km • Years of experience have led biochemists to a general conclusion: • For its preferred substrate, the Km value of an enzyme is usually within a factor of 50 of the steady-state concentration of that substrate. • So if we find that Km = 0.2 mM for the primary substrate of an enzyme, then we expect that the steady-state concentration of that substrate is between 4 µM and 10 mM. Biochem: Inhibition & Mechanisms

  7. Example:hexokinase isozymes Mutant human type I hexokinase PDB 1DGK, 2.8Å110 kDa monomer • Hexokinase catalyzeshexose + ATP  hexose-6-P + ADP • Most isozymes of hexokinase prefer glucose; some also work okay mannose and fructose • Muscle hexokinases have Km ~ 0.1mM so they work efficiently in blood, where [glucose] ~ 4 mM • Liver glucokinase has Km = 10 mM, which is around the liver [glucose] and can respond to fluctuations in liver [glucose] Biochem: Inhibition & Mechanisms

  8. L-B plots for ordered sequential reactions • http://www-biol.paisley.ac.uk/kinetics/Chapter_4/chapter4_3.html • Plot 1/v0 vs. 1/[A] for various [B] values;flatter slopes correspond to larger [B] • Lines intersect @ a pointin between X intercept and Y intercept Biochem: Inhibition & Mechanisms

  9. L-B plots for ping-pong reactions • Again we plot 1/v vs 1/[A] for various [B] • Parallel lines (same kcat/Km);lower lines correspond to larger [B] • http://www-biol.paisley.ac.uk/kinetics/Chapter_4/chapter4_3_2.html Biochem: Inhibition & Mechanisms

  10. Inhibition is important both conceptually and practically • We study inhibition to clarify our understanding of enzyme mechanisms and because knowing how inhibition works helps us design pharmaceuticals. Biochem: Inhibition & Mechanisms

  11. Why study inhibition? • Let’s look at how enzymes get inhibited. • At least two reasons to do this: • We can use inhibition as a probe for understanding the kinetics and properties of enzymes in their uninhibited state; • Many—perhaps most—drugs are inhibitors of specific enzymes. • We'll see these two reasons for understanding inhibition as we work our way through this topic. Biochem: Inhibition & Mechanisms

  12. The concept of inhibition • An enzyme is a biological catalyst, i.e. a substance that alters the rate of a reaction without itself becoming permanently altered by its participation in the reaction. • The ability of an enzyme (particularly a proteinaceous enzyme) to catalyze a reaction can be altered by binding small molecules to it: • sometimes at its active site • sometimes at a site distant from the active site. Biochem: Inhibition & Mechanisms

  13. Inhibitors and accelerators • Usually these alterations involve a reduction in the enzyme's ability to accelerate the reaction; less commonly, they give rise to an increase in the enzyme's ability to accelerate a reaction. Biochem: Inhibition & Mechanisms

  14. Why more inhibitors than accelerators? • Natural selection: if there were small molecules that can facilitate the enzyme's propensity to speed up a reaction, nature probably would have found a way to incorporate those facilitators into the enzyme over the billions of years that the enzyme has been available. • Most enzymes are already fairly close to optimal in their properties; we can readily mess them up with effectors, but it's more of a challenge to find ways to make enzymes better at their jobs. Biochem: Inhibition & Mechanisms

  15. Types of inhibitors • Irreversible • Inhibitor binds without possibility of release • Usually covalent • Each inhibition event effectively removes a molecule of enzyme from availability • Reversible • Usually noncovalent (ionic or van der Waals) • Several kinds • Classifications somewhat superseded by detailed structure-based knowledge of mechanisms, but not entirely Biochem: Inhibition & Mechanisms

  16. Types of reversible inhibition • Competitive • Inhibitor binds at active site • Prevents binding of substrate • Noncompetitive • Inhibitor binds distant from active site • Interferes with turnover • Uncompetitive (rare?) • Inhibitor binds to ES complex • Removes ES, interferes with turnover • Mixed(usually Competitive + Noncompetitive) Biochem: Inhibition & Mechanisms

  17. Putting that all together… Ligands that influence enzyme activity Accelerators Inhibitors (Usually allosteric) Irreversible Reversible (Usually covalent) Competitive Mixed Noncompetitive Uncompetitive Biochem: Inhibition & Mechanisms

  18. How to tell them apart • Reversible vs irreversible • dialyze an enzyme-inhibitor complex against a buffer free of inhibitor • if turnover or binding still suffers, it’s irreversible • Competitive vs. other reversible: • Structural studies if feasible • Kinetics Biochem: Inhibition & Mechanisms

  19. Competitive inhibition • Put in a lot of substrate:ability of the inhibitor to getin the way of the binding is hindered:out-competed by sheer #s of substrate molecules. • This kind of inhibition manifests itself as interference with binding, i.e. with an increase of Km Biochem: Inhibition & Mechanisms

  20. Competitive inhibitors don’t affect turnover • Within the active site of any given molecule of the enzyme, one of three states are possible: • The inhibitor is present • The substrate is present • Nothing is present • Therefore the rate of turnover isn’t affected by the inhibitor: just the availability of binding sites. Biochem: Inhibition & Mechanisms

  21. Kinetics of competition • Competitive inhibitor hinders binding of substrate but not reaction velocity: • Affects the Km of the enzyme, not Vmax. • Which way does it affect it? • Km = amount of substrate that needs to be present to run the reaction velocity up to half its saturation velocity. • Competitive inhibitor requires us to shove more substrate into the reaction in order to achieve that half-maximal velocity. • So: competitive inhibitor increasesKm Biochem: Inhibition & Mechanisms

  22. L-B: competitive inhibitor • Km goes up so -1/ Km moves toward origin • Vmax unchanged so Y intercept unchanged Biochem: Inhibition & Mechanisms

  23. Competitive inhibitor:Quantitation of Ki • Define inhibition constantKi to be the concentration of inhibitor that increases Km by a factor of two. • Then Km,obs = Km(1+[Ic]/Ki) • So [Ic] that moves Km halfway to the origin is Ki. • If Ki = 100 nM and [Ic] = 1 µM, then we’ll increase Km,obs elevenfold! Biochem: Inhibition & Mechanisms

  24. Noncompetitive inhibition S I • Noncompetitive inhibitor has no influence on how available the binding site for substrate is, so it doesn’t affect Km at all • However, it has a profound inhibitory influence on the speed of the reaction, i.e. turnover. So it reducesVmax and has no influence on Km. Biochem: Inhibition & Mechanisms

  25. L-B for non-competitives • Decrease in Vmax 1/Vmax is larger • X-intercept unaffected Biochem: Inhibition & Mechanisms

  26. Ki for noncompetitives • Ki defined as concentration of inhibitor that cuts Vmax in half • Vmax,obs =Vmax/(1 + [In]/Ki) • In previous figure the “high” concentration of inhibitor is Ki • If Ki = Ki’, this is pure noncompetitive inhibition Biochem: Inhibition & Mechanisms

  27. Uncompetitive inhibition • Inhibitor binds only if ES has already formed • It creates a ternary ESI complex • This removes ES, so by LeChatlier’s Principle it actually drives the original reaction (E + S  ES) to the right; so it decreasesKm • But it interferes with turnover so Vmax goes down • If Km and Vmax decrease at the same rate, then it’s classical uncompetitive inhibition. Biochem: Inhibition & Mechanisms

  28. L-B for uncompetitives • Km moves away from the origin • Vmax moves away from the origin • Slope (Km/Vmax) is unchanged Biochem: Inhibition & Mechanisms

  29. Ki for uncompetitives • Defined as inhibitor concentration that cuts Vmax or Km in half • Easiest to read from Vmax value • Iu labeled “high” is Ki in this plot Biochem: Inhibition & Mechanisms

  30. iClicker question 3 What assumptions does the standard derivation of the Michaelis-Menten equation depend on? • (a) d[ES]/dt = 0 for some reasonable period • (b) Formation of product is rate-limiting • (c) [P] = 0 at time t=0, or else k-2 = 0 • (d) All of the above • (e) None of the above Biochem: Inhibition & Mechanisms

  31. iClicker quiz, question 4 Treatment of enzyme E with compound Y doubles Km and leaves Vmax unchanged. Compound Y is: • (a) an accelerator of the reaction • (b) a competitive inhibitor • (c) a non-competitive inhibitor • (d) an uncompetitive inhibitor Biochem: Inhibition & Mechanisms

  32. iClicker quiz, question 5 Treatment of enzyme E with compound X doubles Vmax and leaves Km unchanged. Compound X is: • (a) an accelerator of the reaction • (b) a competitive inhibitor • (c) a non-competitive inhibitor • (d) an uncompetitive inhibitor Biochem: Inhibition & Mechanisms

  33. Mixed inhibition • Usually involves interference with both binding and catalysis • Km goes up, Vmax goes down • Easy to imagine the mechanism: • Binding of inhibitor alters the active-site configuration to interfere with binding, but it also alters turnover • Same picture as with pure noncompetitive inhibition, but with Ki ≠ Ki’ Biochem: Inhibition & Mechanisms

  34. Most pharmaceuticals are enzyme inhibitors • Some are inhibitors of enzymes that are necessary for functioning of pathogens • Others are inhibitors of some protein whose inappropriate expression in a human causes a disease. • Others are targeted at enzymes that are produced more energetically by tumors than they are by normal tissues. Biochem: Inhibition & Mechanisms

  35. Characteristics of Pharmaceutical Inhibitors • Usually competitive, i.e. they raise Km without affecting Vmax • Some are mixed, i.e. Km up, Vmax down • Iterative design work will decrease Kifrom millimolar down to nanomolar • Sometimes design work is purely blind HTS; other times, it’s structure-based Biochem: Inhibition & Mechanisms

  36. Amprenavir • Competitive inhibitor of HIV protease,Ki = 0.6 nM for HIV-1 • No longer sold: mutual interference with rifabutin, which is an antibiotic used against a common HIV secondary bacterial infection, Mycobacterium avium Biochem: Inhibition & Mechanisms

  37. When is a good inhibitor a good drug? • It needs to be bioavailable and nontoxic • Beautiful 20nM inhibitor is often neither • Modest sacrifices of Ki in improving bioavailability and non-toxicity are okay if Ki is low enough when you start sacrificing Biochem: Inhibition & Mechanisms

  38. How do we lessen toxicity and improve bioavailability? • Increase solubility…that often increases Ki because the van der Waals interactions diminish • Solubility makes it easier to get the compound to travel through the bloodstream • Toxicity is often associated with fat storage, which is more likely with insoluble compounds Biochem: Inhibition & Mechanisms

  39. Drug-design timeline 100 -3 • 2 years of research, 8 years of trials Improving affinity Toxicity and bioavailability Stage II clinical trials Cost/yr, 106 $ Stage I clinical trials Preliminary toxicity testing log Ki -8 10 Research Clinical Trials 0 2 Time, Yrs 10 Biochem: Inhibition & Mechanisms

  40. Atomic-Level Mechanisms • We want to understand atomic-level events during an enzymatically catalyzed reaction. • Sometimes we want to find a way to inhibit an enzyme • in other cases we're looking for more fundamental knowledge, viz. the ways that biological organisms employ chemistry and how enzymes make that chemistry possible. Biochem: Inhibition & Mechanisms

  41. How we study mechanisms • There are a variety of experimental tools available for understanding mechanisms, including isotopic labeling of substrates, structural methods, and spectroscopic kinetic techniques. Biochem: Inhibition & Mechanisms

  42. Overcoming the barrier • Simple system:single high-energy transition state intermediate between reactants, products Free Energy G‡ R P Reaction Coordinate Biochem: Inhibition & Mechanisms

  43. Intermediates • Often there is a quasi-stable intermediate state midway between reactants & products; transition states on either side T2 T1 Free Energy I R P Reaction Coordinate Biochem: Inhibition & Mechanisms

  44. Activation energy & temperature • It’s intuitively sensible that higher temperatures would make it easier to overcome an activation barrier • Rate k(T) = Q0exp(-G‡/RT) • G‡ = activation energy or Arrhenius energy • This provides tool for measuring G‡ Svante Arrhenius Biochem: Inhibition & Mechanisms

  45. Determining G‡ • Rememberk(T) = Q0exp(-G‡/RT) • ln k = lnQ0 - G‡/RT • Measure reaction rate as function of temperature • Plot ln k vs 1/T; slope will be -G‡/R catalyzed ln k uncatalyzed 1/T, K-1 Biochem: Inhibition & Mechanisms

  46. How enzymes alter G‡ • Enzymes reduce DG‡ by allowing the binding of the transition state into the active site • Binding of the transition state needs to be tighter than the binding of either the reactants or the products. Biochem: Inhibition & Mechanisms

  47. DG‡ and Entropy • Effect is partly entropic: • When a substrate binds,it loses a lot of entropy. • Thus the entropic disadvantage of (say) a bimolecular reaction is soaked up in the process of binding the first of the two substrates into the enzyme's active site. Biochem: Inhibition & Mechanisms

  48. Enthalpy and transition states • Often an enthalpic component to the reduction in DG‡ as well • Ionic or hydrophobic interactions between the enzyme's active site residues and the components of the transition state make that transition state more stable. Biochem: Inhibition & Mechanisms

  49. Reactants bound by enzyme are properly positioned Get into transition-state geometry more readily Transition state is stabilized Two ways to change G‡ AB AB E E A+B A+B A-B A-B Biochem: Inhibition & Mechanisms

  50. How do enzymes reduce activation energies? • We can illustrate mechanistic principles by looking at specific examples; we can also recognize enzyme regulation when we see it. Biochem: Inhibition & Mechanisms

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