Unlocking Enzyme Inhibition: Mechanisms & Significance

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