Enzyme Inhibition

1. Enzyme Inhibition Andy HowardIntroductory Biochemistry 12 November 2014 Enzyme Inhibition & Mechanisms

2. What we’ll discuss • Exchange Reactions and Kinetics • Inhibition: General • Irreversible & reversible inhibition • Types of reversible inhibition • Kinetics of inhibition • Pharmaceutical inhibitors Enzyme Inhibition & Mechanisms

3. Using exchange reactions to discern mechanisms • Example: sucrose phosphorylase and maltose phosphorylase both cleave disaccharides and add Pi to one product: • Sucrose + Pi glucose-1-P + fructose • Maltose + Pi glucose-1-P + glucose • Try 32P tracers with G-1-P:G-1-P + 32Pi Pi + G-1-32Pi • … so what happens with these two enzymes? Enzyme Inhibition & Mechanisms

4. Sucrose & maltose phosphorylase • Sucrose phosphorylase doescatalyze the exchange;not maltose phosphorylase • This suggests that SucPase usesdouble-displacement reaction;MalPase uses a single-displacement • Sucrose + E  E-glucose + fructoseE-glucose + Pi E + glucose-1-P • Maltose + E + Pi Maltose:E:PiMaltose:E:Pi glucose-1P + glucose Sucrose phosphorylaseBifidobacterium113 kDa dimerPDB 1R7A, 1.77ÅEC 2.4.1.7 Enzyme Inhibition & Mechanisms

5. 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. Enzyme Inhibition & Mechanisms

6. 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 Enzyme Inhibition & Mechanisms

7. 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. Enzyme Inhibition & Mechanisms

8. 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. Enzyme Inhibition & Mechanisms

9. 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) Enzyme Inhibition & Mechanisms

10. Types of inhibitors (G&G §13.4) • 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 Enzyme Inhibition & Mechanisms

11. 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 • Characterized by KI KI’ Enzyme Inhibition & Mechanisms

12. 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 Enzyme Inhibition & Mechanisms

13. 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 Enzyme Inhibition & Mechanisms

14. 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. Enzyme Inhibition & Mechanisms

15. 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 Enzyme Inhibition & Mechanisms

16. L-B: pure competitive inhibitor (G&G Fig. 13.13) • Km goes up so -1/ Km moves toward origin • Vmax unchanged so Y intercept unchanged Enzyme Inhibition & Mechanisms

17. Competitive inhibitor:Quantitation of Ki • Define inhibition constant Ki to be the concentration of inhibitor that increases Km by a factor of two. • 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! Enzyme Inhibition & Mechanisms

18. Think about that equation! • Remember that it says Km,obs = Km{1 + ([Ic]/Ki)} • It does NOT say Km,obs = Km{(1+[Ic])/Ki} • … which would be nonsensical because [Ic] has dimensions and 1 doesn’t • In fact, Ic and Ki have the same dimensions,so they cancel like they should! • But every year several students get that wrong. Don’t be among them! Enzyme Inhibition & Mechanisms

19. Don’t get lazy! • A competitive inhibitor doesn’t automatically double Km • The amount by which the inhibitor increases Km is dependent on [I]c • If it happens that [Ic] = KI, then Km will double, as the equation shows Enzyme Inhibition & Mechanisms

20. Noncompetitive inhibition S I • Inhibitor binds distant fromactive site, so it binds to theenzyme whether the substrateis present or absent. • Noncompetitive inhibitor has no influence on how available the binding site for substrate is, so it does not 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. Enzyme Inhibition & Mechanisms

21. L-B for pure non-competitive inhibitors • Decrease in Vmax 1/Vmax is larger • X-intercept unaffected Cf. G&G Fig. 13.15 Enzyme Inhibition & Mechanisms

22. 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 Enzyme Inhibition & Mechanisms

23. Same warning as before . . . • Correct: Vmax,obs = Vmax/{1 + ([In]/Ki)} • Incorrect: Vmax,obs = Vmax/{(1 + [In])/Ki} • As in the previous instance, the incorrect formula makes no sense because [In] has dimensions and 1 doesn’t. Enzyme Inhibition & Mechanisms

24. Uncompetitive inhibition • Inhibitor binds only if ES has already formed • It creates a ternary ESI complex • This removes ES, so by LeChatelier’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. Enzyme Inhibition & Mechanisms

25. L-B for uncompetitives • -1/Km moves away from origin • 1/Vmax moves away from the origin • Slope (Km/Vmax) is unchanged Cf. G&G fig. 13.17 Enzyme Inhibition & Mechanisms

26. Ki for uncompetitives • Defined as inhibitor concentration that cuts Vmax or Km in half • Easiest to read from Vmax value • Vmax,obs = Vmax/{1+([I]u/KI)} • Iu labeled “high” is Ki in this plot Enzyme Inhibition & Mechanisms

27. iClicker quiz, question 1 1. Treatment of enzyme E with compound Y doubles Km and leaves Vmaxunchanged. Compound Y is: • (a) an accelerator of the reaction • (b) a competitive inhibitor • (c) a non-competitive inhibitor • (d) an uncompetitive inhibitor Enzyme Inhibition & Mechanisms

28. iClicker quiz, question 2 2. 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 Enzyme Inhibition & Mechanisms

29. 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’ Cf. G&G fig. 13.16 Enzyme Inhibition & Mechanisms

30. Summary: reversible inhibitors Enzyme Inhibition & Mechanisms

31. 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. Enzyme Inhibition & Mechanisms

32. 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 Enzyme Inhibition & Mechanisms

33. 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 Enzyme Inhibition & Mechanisms

34. 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 Enzyme Inhibition & Mechanisms

35. 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 Enzyme Inhibition & Mechanisms

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