1. 10/28/2010Enzyme inihibition, mechanisms Enzymes: inhibition and mechanisms Andy Howard Introductory Biochemistry 28 October 2010

2. 10/28/2010 Enzyme inihibition, mechanisms P. 2 of 44 Inhibition and mechanisms Inhibition is important in its own right and as a tool for understanding kinetics and mechanisms

3. 10/28/2010 Enzyme inihibition, mechanisms P. 3 of 44 Inhibition & Mechanism Topics Inhibitors Types of inhibitors Kinetics of inhibition Pharmaceuticals What makes an inhibitor a useful drug? Mechanisms Terminology Activation barrier E-X* complexes Enthalpy & entropy Protein Motion

4. 10/28/2010 Enzyme inihibition, mechanisms P. 4 of 44 Distinctions we can make Inhibitors can be reversible or irreversible Where do they bind? At the enzyme’s active site At a site distant from the active site. To what do they bind? To the unliganded enzyme E To the enzyme-intermediate complex or the enzyme-substrate complex (ES) To both (E or ES)

5. 10/28/2010 Enzyme inihibition, mechanisms P. 5 of 44 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enzyme from availability Reversible Usually noncovalent (ionic or van der Waals) Several kinds Classifications somewhat superseded by detailed structure-based knowledge of mechanisms, but not entirely

6. 10/28/2010 Enzyme inihibition, mechanisms P. 6 of 44 Types of reversible inhibition Competitive Inhibitor binds at active site of unliganded enzyme Prevents binding of substrate Noncompetitive Inhibitor binds distant from active site (E or ES) Interferes with turnover Uncompetitive (rare?) Inhibitor binds only to ES complex Removes ES, interferes with turnover Mixed (usually Competitive + Noncompetitive)

7. 10/28/2010 Enzyme inihibition, mechanisms P. 7 of 44 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

8. 10/28/2010 Enzyme inihibition, mechanisms P. 8 of 44 Competitive inhibition Put in a lot of substrate: ability of the inhibitor to get in 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 K m

9. 10/28/2010 Enzyme inihibition, mechanisms P. 9 of 44 Competitive inhibitors don’t affect turnover If the substrates manages to bind even though there is inhibitor present, then it can be turned over just as quickly as if the inhibitor is absent; so the inhibitor influences binding but not turnover.

10. 10/28/2010 Enzyme inihibition, mechanisms P. 10 of 44 Kinetics of competition Competitive inhibitor hinders binding of substrate but not reaction velocity: Affects the K m of the enzyme, not V max. Which way does it affect it? K m = 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 increases K m

11. 10/28/2010 Enzyme inihibition, mechanisms P. 11 of 44 L-B: competitive inhibitor K m goes up so -1/ K m moves toward origin V max unchanged so Y intercept unchanged

12. 10/28/2010 Enzyme inihibition, mechanisms P. 12 of 44 Competitive inhibitor: Quantitation of K i Define inhibition constant K i to be the concentration of inhibitor that increases K m by a factor of two. K m,obs = K m (1+[ I c ]/K i ) So [ I c ] that moves K m halfway to the origin is K i. If K i = 100 nM and [ I c ] = 1 µM, then we’ll increase K m,obs elevenfold!

13. 10/28/2010 Enzyme inihibition, mechanisms P. 13 of 44 Think about that equation! Remember that it says K m,obs = K m (1+[ I c ]/K i ) It does NOT say K m,obs = K m [(1+[ I c ])/K i ] … which would be nonsensical because [ I c ] has dimensions and 1 doesn’t In fact, I c and K i have the same dimensions, so they cancel like they should! But every year several students get that wrong. Don’t be among them!

14. 10/28/2010 Enzyme inihibition, mechanisms P. 14 of 44 Don’t get lazy! A competitive inhibitor doesn’t automatically double K m The amount by which the inhibitor increases K m is dependent on [I] c If it happens that [I] c = K I, then K m will double, as the equation shows

15. 10/28/2010 Enzyme inihibition, mechanisms P. 15 of 44 Noncompetitive inhibition Inhibitor binds distant from active site, so it binds to the enzyme whether the substrate is present or absent. Noncompetitive inhibitor has no influence on how available the binding site for substrate is, so it does not affect K m at all However, it has a profound inhibitory influence on the speed of the reaction, i.e. turnover. So it reduces V max and has no influence on K m. S I

16. 10/28/2010 Enzyme inihibition, mechanisms P. 16 of 44 L-B for non-competitives Decrease in V max  1/V max is larger X-intercept unaffected

17. 10/28/2010 Enzyme inihibition, mechanisms P. 17 of 44 K i for noncompetitives K i defined as concentration of inhibitor that cuts V max in half V max,obs = V max /(1 + [ I n ]/K i ) In previous figure the “high” concentration of inhibitor is K i If K i = K i ’, this is pure noncompetitive inhibition

18. 10/28/2010 Enzyme inihibition, mechanisms P. 18 of 44 Same warning as before... Correct: V max,obs = V max /(1 + [ I n ]/K i ) Incorrect: V max,obs = V max /[(1 + [ I n ])/K i ] As in the previous instance, the incorrect formula makes no sense because [ I n ] has dimensions and 1 doesn’t.

19. 10/28/2010 Enzyme inihibition, mechanisms P. 19 of 44 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 decreases K m But it interferes with turnover so V max goes down If K m and V max decrease at the same rate, then it’s classical uncompetitive inhibition.

20. 10/28/2010 Enzyme inihibition, mechanisms P. 20 of 44 L-B for uncompetitives -1/K m moves away from origin 1/V max moves away from the origin Slope (  K m /V max ) is unchanged

21. 10/28/2010 Enzyme inihibition, mechanisms P. 21 of 44 K i for uncompetitives Defined as inhibitor concentration that cuts V max or K m in half Easiest to read from V max value V max,obs = V max /(1+[I] u /K I ) I u labeled “high” is K i in this plot

22. 10/28/2010 Enzyme inihibition, mechanisms P. 22 of 44 iClicker quiz, question 1 1. Treatment of enzyme E with compound Y doubles K m and leaves V max unchanged. Compound Y is: (a) an accelerator of the reaction (b) a competitive inhibitor (c) a non-competitive inhibitor (d) an uncompetitive inhibitor

23. 10/28/2010 Enzyme inihibition, mechanisms P. 23 of 44 iClicker quiz, question 2 2. Treatment of enzyme E with compound X doubles V max and leaves K m unchanged. Compound X is: (a) an accelerator of the reaction (b) a competitive inhibitor (c) a non-competitive inhibitor (d) an uncompetitive inhibitor

24. 10/28/2010 Enzyme inihibition, mechanisms P. 24 of 44 Mixed inhibition Usually involves interference with both binding and catalysis K m goes up, V max 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 K i ≠ K i ’

25. 10/28/2010 Enzyme inihibition, mechanisms P. 25 of 44 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.

26. 10/28/2010 Enzyme inihibition, mechanisms P. 26 of 44 Characteristics of Pharmaceutical Inhibitors Usually competitive, i.e. they raise K m without affecting V max Some are mixed, i.e. K m up, V max down Iterative design work will decrease K i from millimolar down to nanomolar Sometimes design work is purely blind HTS; other times, it’s structure-based

27. 10/28/2010 Enzyme inihibition, mechanisms P. 27 of 44 Amprenavir Competitive inhibitor of HIV protease, K i = 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

28. 10/28/2010 Enzyme inihibition, mechanisms P. 28 of 44 When is a good inhibitor a good drug? It needs to be bioavailable and nontoxic Beautiful 20nM inhibitor is often neither Modest sacrifices of K i in improving bioavailability and non-toxicity are okay if K i is low enough when you start sacrificing

29. 10/28/2010 Enzyme inihibition, mechanisms P. 29 of 44 How do we lessen toxicity and improve bioavailability? Increase solubility… that often increases K i 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

30. 10/28/2010 Enzyme inihibition, mechanisms P. 30 of 44 Drug-design timeline 2 years of research, 8 years of trials log K i Time, Yrs10 20 -3 -8 Cost/yr, 10 6 $ 10 100 Improving affinity Toxicity and bioavailability Research Clinical Trials Preliminary toxicity testing Stage I clinical trials Stage II clinical trials

31. 10/28/2010 Enzyme inihibition, mechanisms P. 31 of 44 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.

32. 10/28/2010 Enzyme inihibition, mechanisms P. 32 of 44 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.

33. 10/28/2010 Enzyme inihibition, mechanisms P. 33 of 44 Overcoming the barrier Simple system: single high-energy transition state intermediate between reactants, products Free Energy Reaction Coordinate R P G‡G‡

34. 10/28/2010 Enzyme inihibition, mechanisms P. 34 of 44 Intermediates Often there is a quasi-stable intermediate state midway between reactants & products; transition states on either side Free Energy R P T1 T2 I Reaction Coordinate

35. 10/28/2010 Enzyme inihibition, mechanisms P. 35 of 44 Activation energy & temperature It’s intuitively sensible that higher temperatures would make it easier to overcome an activation barrier Rate k(T) = Q 0 exp(-  G ‡ /RT)  G ‡ = activation energy or Arrhenius energy This provides tool for measuring  G ‡ Svante Arrhenius

36. 10/28/2010 Enzyme inihibition, mechanisms P. 36 of 44 Determining  G ‡ Remember k(T) = Q 0 exp(-  G ‡ /RT) ln k = lnQ 0 -  G ‡ /RT Measure reaction rate as function of temperature Plot ln k vs 1/T; slope will be -  G ‡ /R ln k 1/T, K -1 uncatalyzed catalyzed

37. 10/28/2010 Enzyme inihibition, mechanisms P. 37 of 44 How enzymes alter  G ‡ Enzymes reduce  G ‡ 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. In fact, the enzyme must stabilize the transition-state complex EX ‡ more than it stabilizes the substrate complex ES (see section 14.2).

38. 10/28/2010 Enzyme inihibition, mechanisms P. 38 of 44 Dissociation constants for ES and EX* Dissociation constant for ES: K s = [E][S]/[ES] Dissociation constant for EX ‡ : K T = [E][X ‡ ]/[EX ‡ ] Transition state theory says the ratio of reaction rates is related to the ratio of these: k e /k u = K s / K T

39. 10/28/2010 Enzyme inihibition, mechanisms P. 39 of 44 What makes EX ‡ more stable than ES? Intrinsic (enthalpic) binding energy of ES makes it a lower-energy species than E+S; but we want EX* to be lower. ES loses entropy relative to E + S ES is sometimes strained, distorted, or desolvated relative to E+S So if EX ‡ is less strained and has more entropy, we win See section 14.3

40. 10/28/2010 Enzyme inihibition, mechanisms P. 40 of 44 How tight is the binding? Section 14.4 gives some examples Transition-state analogs are stable molecules that are geometrically and electrostatically similar to transition states Sometimes the analogs bind ~ 160 - 40000 times more avidly than substrates 1,6-hydrate of purine nucleoside binds to adenosine deaminase with K I = 3*10 -13 M

41. 10/28/2010 Enzyme inihibition, mechanisms P. 41 of 44  G ‡ 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.

42. 10/28/2010 Enzyme inihibition, mechanisms P. 42 of 44 Enthalpy and transition states Often an enthalpic component to the reduction in  G ‡ 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.

43. 10/28/2010 Enzyme inihibition, mechanisms P. 43 of 44 Two ways to change  G ‡ Reactants bound by enzyme are properly positioned Get into transition- state geometry more readily Transition state is stabilized E ABAB E ABAB A+B A-B

44. 10/28/2010 Enzyme inihibition, mechanisms P. 44 of 44 The protein moves as well! Changes to active-site conformation: Help with substrate binding Position the catalytic groups Induce formation of an NAC Help to break or make bonds Facilitate conversion of S to P Sometimes involve networks of concerted amino acid changes