Enzyme Mechanisms: Understanding Activation Energy Reduction

1. Enzyme Mechanisms Andy HowardBiochemistry Lectures, Spring 201919 February 2019

2. Mechanisms matter • Today’s inquiry is the mechanisms by which enzymes reduce activation energy barriers. • We’ll offer some examples of these mechanisms. Mechanisms, Examples

3. Mechanisms Transition states Binding modes Proximity Transition-state stabilization Chemical modes Acid-base Covalent catalysis Mechanisms, concluded Induced fit Participating moieties Serine proteases Other mechanisms Topics for today Mechanisms, Examples

4. 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. Mechanisms, Examples

5. 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. Mechanisms, Examples

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

7. 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 Mechanisms, Examples

8. 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 Mechanisms, Examples

9. 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. Mechanisms, Examples

10. 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. Mechanisms, Examples

11. 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. Mechanisms, Examples

12. 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 Mechanisms, Examples

13. Binding and Chemistry • Catalysis via binding • Proximity effect • Transition state stabilization • Catalysis via chemistry • Acid-base reactions • Covalent catalysis Mechanisms, Examples

14. Binding modes: proximity • We describe enzymatic mechanisms in terms of the binding modes of the substrates (or, more properly, the transition-state species) to the enzyme. William Jencks Mechanisms, Examples

15. Proximity effect One of these involves the proximity effect, in which two (or more) substrates are directed down potential-energy gradients to positions where they are close to one another. Thus the enzyme is able to defeat the entropic difficulty of bringing substrates together. Mechanisms, Examples

16. Binding modes: efficient transition-state binding • Transition state fits even better (geometrically and electrostatically) in the active site than the substrate would. This improved fit lowers the energy of the transition-state system relative to the substrate. • Best competitive inhibitors of an enzyme are those that resemble the transition state rather than the substrate or product. Mechanisms, Examples

17. Examples illustrating transition state stabilization • Numerous enzymes act by providing stabilization of a transition state or an intermediate • Giveaway is extremely effective competitive inhibitors that resemble the transition state that is being stabilized Mechanisms, Examples

18. Adenosine deaminase with transition-state analog • Transition-state analog:Ki~10-8 * substrate Km • Wilson et al (1991) Science252: 1278 Mechanisms, Examples

19. ADA transition-state analog • 1,6 hydrate of purine ribonucleoside binds with KI ~ 3*10-13 M Mechanisms, Examples

20. Diffusion-controlled reactions • Some enzymes are so efficient that the limiting factor in completion of the reaction is diffusion of the substrates into the active site: • These are diffusion-controlled reactions. • Ultra-high turnover rates: kcat ~ 108 s-1. • We can describe kcat / Km as catalytic efficiency of an enzyme. A diffusion-controlled reaction will have a catalytic efficiency on the order of 108 M-1s-1. Mechanisms, Examples

21. Induced fit (CF&M fig. 6.4) • Refinement on original Emil Fischer lock-and-key notion: • both the substrate (or transition-state) and the enzyme have flexibility • Binding induces conformational changes Mechanisms, Examples

22. Ionic reactions (CF&M§7.6) • Define them as reactions that involve charged, or at least polar, intermediates • Typically 2 reactants • Electron rich (nucleophilic) reactant • Electron poor (electrophilic) reactant Mechanisms, Examples

23. Describing nucleophilic substitutions Conventional to describe reaction as attack of nucleophile on electrophile Drawn with nucleophile donating electron(s) to electrophile Mechanisms, Examples

24. Attack on Acyl Group • Transfer of an acyl group • Nucleophile Y attacks carbonyl carbon, forming tetrahedral intermediate • X- is leaving group Mechanisms, Examples

25. Direct Displacement • Attacking group adds to face of atom opposite to leaving group • Transition state has five ligands;inherently less stable than schemes involving only four ligands on a carbon; but they still play out (Moran eqn. 6.2) Mechanisms, Examples

26. Cleavage Reactions • Both electrons stay with one atom • Covalent bond produces carbanion:R3—C—H  R3—C:- + H+ • Covalent bond produces carbocation:R3—C—H  R3—C+ + :H- • One electron stays with each product • Both end up as radicals • R1O—OR2 R1O• + •OR2 • Radicals are reactive— some more than others Mechanisms, Examples

27. Covalent catalysis • Refers to situations where a protein side-chain becomes directly involved in a (possibly unstable) covalent bond with one of the reactive species • Example: serine proteases: see beginning of next lecture Mechanisms, Examples

28. Groups of amino acids • An enzyme generally has two or three absolutely critical side-chains that are directly involved in catalysis • Specific examples discussed in section 6.4 of your text: attend to those! • Diads: Arg-arg, carboxylate-carboxylate, carboxylate-histidine Mechanisms, Examples

29. Metal ions in catalysis • Alkalai & alkaline earth: loosely bound, primarily playing structural roles • Transition metals are often directly involved in catalysis, sometimes as Lewis acids • Some transition metals can get involved in redox reactions too Mechanisms, Examples

30. Coenzymes • We’ll go into this in greater detail later • Many are vitamins or are derived (via simple metabolic conversions) from vitamins • Two categories: • Cosubstrates (loosely bound, recycled) • Prosthetic groups (tightly bound; restored to starting state in situ) Mechanisms, Examples

31. Temperature in enzymatic reactions • Earlier discussion of Arrhenius: warmer = faster • If the enzyme unfolds, it can’t catalyze the reaction at all • So there’s an optimum temperature for any particular enzyme • In homeothermic organisms the internal temperature is regulated anyway Mechanisms, Examples

32. pH as an influence • Often there are two or three ionizable groups involved in a catalytic reaction • Both (or all three) need to be in the correct protonation state in order for the reaction to proceed • That sometimes imposes a specific optimal pH for maximal enzyme activity Mechanisms, Examples

33. Oxidation-Reduction Reactions • Commonplace in biochemistry: EC 1 • Oxidation is a loss of electrons • Reduction is the gain of electrons Mechanisms, Examples

34. Analyzing redox reactions • In practice, often: • oxidation is decrease in # of C-H bonds; • reduction is increase in # of C-H bonds • Mnemonic: OIL RIG • Oxidation is loss of electrons • Reduction is gain of electrons Mechanisms, Examples

35. Redox, continued • Intermediate electron acceptors and donors are organic moieties or metals • Ultimate electron acceptor in aerobic organisms is usually dioxygen (O2) • Anaerobic organisms usually employ other electron acceptors (Fe3+, S, …) Mechanisms, Examples

36. Biological redox reactions I • Generally, but not always,these are 2-electron transformations • Often involve alcohols, aldehydes, ketones, carboxylic acids, C=C bonds: • R1R2CH-OH + X  R1R2C=O + XH2 • R1HC=O + X + OH- R1COO- + XH2 • X is usually NAD, NADP, FAD, FMN • A few biological redox systems involve metal ions or Fe-S complexes Mechanisms, Examples

37. Biological Redox Reactions II • FAD and FMN can operate one electron at a time, because they produce a quasi-stable free radical species in the middle • Metal ion-mediated redox systems usually operate one electron at a time Mechanisms, Examples

38. Redox and energy Usually reduced compounds are higher-energy than the corresponding oxidized compounds; therefore oxidation of reduced cofactors (NADH, NADPH, FADH2) usually releases energy that can be harnessed for some other purpose Mechanisms, Examples

39. Induced fit Daniel Koshland • Conformations of enzymes don't change enormously when they bind substrates, but they do change to some extent. An instance where the changes are fairly substantial is the binding of substrates to kinases. Cartoon from textbookofbacteriology.net Mechanisms, Examples

40. Kinase reactions • unwanted reaction ATP + H-O-H ⇒ ADP + Pi • will compete with the desired reactionATP + R-O-H ⇒ ADP + R-O-P • Kinases minimize the likelihood of this unproductive activity by changing conformation upon binding substrate so that hydrolysis of ATP cannot occur until the binding happens. • Illustrates the importance of the order in which things happen in enzyme function Mechanisms, Examples

41. Example of induced fit: hexokinase • Glucose + ATP  Glucose-6-P + ADP • Risk: unproductive reaction with water • Enzyme exists in open & closed forms • Glucose induces conversion to closed form; water can’t do that • Energy expended moving to closed form Mechanisms, Examples

42. Hexokinase conformational changes G&G Fig. 13.28 Mechanisms, Examples

43. Serine protease mechanism (CF&M §6.5, 7.5) • Only detailed mechanism that we’ll ask you to memorize • One of the first to be elucidated • Well studied structurally • Illustrates many other mechanisms • Instance of convergent and divergent evolution Mechanisms, Examples

44. The reaction • Hydrolytic cleavage of peptide bond • Enzyme usually works on esters too • Found in eukaryotic digestive enzymes and in bacterial systems O CH NH C NH C NH R1 CH O R-1 Mechanisms, Examples

45. How specific are proteases? • Widely-varying substrate specificities • Some proteases are highly specific for particular amino acids at position 1, 2, -1, . . . • Others are more promiscuous • Digestive proteases like chymotrypsin and trypsin are intermediate in specificity Mechanisms, Examples

46. Mechanism • Active-site serine —OH …Without neighboring amino acids, it’s fairly unreactive • becomes powerful nucleophile because OH proton lies near unprotonated N of His • This N can abstract the hydrogen at near-neutral pH • Resulting + charge on His is stabilized by its proximity to a nearby carboxylate group on an aspartate side-chain. Mechanisms, Examples

47. Catalytic triad asp his ser • The catalytic triad of asp, his, and ser is found in an approximately linear arrangement in all the serine proteases, all the way from non-specific, secreted bacterial proteases to highly regulated and highly specific mammalian proteases. Mechanisms, Examples

48. Diagram of first three steps Mechanisms, Examples

49. Diagram of last four steps Diagrams courtesy University of Virginia Mechanisms, Examples

50. Chymotrypsin as example • Catalytic Ser is Ser195 • Asp is 102, His is 57 • Note symmetry of mechanism:steps read similarly L R and R  L Diagram courtesy of Anthony Serianni, University of Notre Dame Mechanisms, Examples