Enzyme Mechanisms & Regulation in Biochemistry: Understanding Activation Energy Reduction

1. Enzyme Mechanisms and Regulation Andy HowardIntroductory Biochemistry, Fall 2010Wednesday 22 September 2010 Biochem:Mechanisms,Regulation

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:Mechanisms,Regulation

3. Mechanisms Binding mode catalysis Chemical catalysis Examples Intermediates Applications of Mechanisms Serine proteases Other proteases Lysozyme Regulation Thermodynamics Enzyme availability Allostery, revisited Regulation of Globins Mechanism & Regulation Topics Biochem:Mechanisms,Regulation

4. 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:Mechanisms,Regulation

5. Examining enzyme mechanisms will help us understand catalysis • Examining general principles of catalytic activity and looking at specific cases will facilitate our appreciation of all enzymes. • We can distinguish between binding-mode mechanisms and chemical mechanisms; we’ll look at both Biochem:Mechanisms,Regulation

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

7. 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. Biochem:Mechanisms,Regulation

8. 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 ~ 109 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. Biochem:Mechanisms,Regulation

9. Induced fit • Refinement on original Emil Fischer lock-and-key notion: • both the substrate (or transition-state) and the enzyme have flexibility • Binding induces conformational changes Biochem:Mechanisms,Regulation

10. Ionic reactions • Define them as reactions that involve charged, or at least polar, intermediates • Typically 2 reactants • Electron rich (nucleophilic) reactant • Electron poor (electrophilic) reactant • Conventional to describe reaction as attack of nucleophile on electrophile • Drawn with nucleophile donating electron(s) to electrophile Biochem:Mechanisms,Regulation

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

12. Direct Displacement • Attacking group adds to face of atom opposite to leaving group (scheme 6.2) • Transition state has five ligands;inherently less stable than scheme 6.1 Biochem:Mechanisms,Regulation

13. 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 highly reactive—some more than others Biochem:Mechanisms,Regulation

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

15. 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 Biochem:Mechanisms,Regulation

16. Biological redox reactions • Generally 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 • Usually reduced compounds are higher-energy than the corresponding oxidized compounds Biochem:Mechanisms,Regulation

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 Biochem:Mechanisms,Regulation

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

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

20. 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 Biochem:Mechanisms,Regulation

21. Hexokinase structure • Diagram courtesy E. Marcotte, UT Austin Biochem:Mechanisms,Regulation

22. Tight binding of ionic intermediates • Quasi-stable ionic species strongly bound by ion-pair and H-bond interactions • Similar to notion that transition states are the most tightly bound species, but these are more stable Biochem:Mechanisms,Regulation

23. Serine protease mechanism • 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 Biochem:Mechanisms,Regulation

24. The reaction • Hydrolytic cleavage of peptide bond • Enzyme usually works on esters too • Found in eukaryotic digestive enzymes and in bacterial systems • Widely-varying substrate specificities • Some proteases are highly specific for particular amino acids at position 1, 2, -1, . . . • Others are more promiscuous O CH NH C NH C NH R1 CH O R-1 Biochem:Mechanisms,Regulation

25. 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. Biochem:Mechanisms,Regulation

26. Catalytic triad • 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. Biochem:Mechanisms,Regulation

27. Diagram of first three steps Biochem:Mechanisms,Regulation

28. Diagram of last four steps Diagrams courtesy University of Virginia Biochem:Mechanisms,Regulation

29. 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 Biochem:Mechanisms,Regulation

30. Oxyanion hole • When his-57 accepts proton from Ser-195:it creates an R—O- ion on Ser sidechain • In reality the Ser O immediately becomes covalently bonded to substrate carbonyl carbon, moving negative charge to the carbonyl O. • Oxyanion is on the substrate's oxygen • Oxyanion stabilized by additional interaction in addition to the protonated his 57:main-chain NH group from gly 193 H-bonds to oxygen atom (or ion) from the substrate,further stabilizing the ion. Biochem:Mechanisms,Regulation

31. Oxyanion hole cartoon • Cartoon courtesy Henry Jakubowski, College of St.Benedict / St.John’s University Biochem:Mechanisms,Regulation

32. Modes of catalysis in serine proteases • Proximity effect: gathering of reactants in steps 1 and 4 • Acid-base catalysis at histidine in steps 2 and 4 • Covalent catalysis on serine hydroxymethyl group in steps 2-5 • So both chemical (acid-base & covalent) and binding modes (proximity & transition-state) are used in this mechanism Biochem:Mechanisms,Regulation

33. Specificity • Active site catalytic triad is nearly invariant for eukaryotic serine proteases • Remainder of cavity where reaction occurs varies significantly from protease to protease. • In chymotrypsin  hydrophobic pocket just upstream of the position where scissile bond sits • This accommodates large hydrophobic side chain like that of phe, and doesn’t comfortably accommodate hydrophilic or small side chain. • Thus specificity is conferred by the shape and electrostatic character of the site. Biochem:Mechanisms,Regulation

34. Chymotrypsin active site • Comfortably accommodates aromatics at S1 site • Differs from other mammalian serine proteases in specificity Diagram courtesy School of Crystallography, Birkbeck College Biochem:Mechanisms,Regulation

35. Divergent evolution • Ancestral eukaryotic serine proteases presumably have differentiated into forms with different side-chain specificities • Chymotrypsin is substantially conserved within eukaryotes, but is distinctly different from elastase • Primary differences are in P1 side chain pocket, but that isn’t inevitable Biochem:Mechanisms,Regulation

36. iClicker quiz, question 1 Why would the nonproductive hexokinase reaction H2O + ATP  ADP + Pibe considered nonproductive? • (a) Because it needlessly soaks up water • (b) Because the enzyme undergoes a wasteful conformational change • (c) Because the energy in the high-energy phosphate bond is unavailable for other purposes • (d) Because ADP is poisonous • (e) None of the above Biochem:Mechanisms,Regulation

37. iClicker Quiz question 2 What would bind tightest in the TIM active site? • (a) DHAP (substrate) • (b) D-glyceraldehyde (product) • (c) 2-phosphoglycolate(Transition-state analog) • (d) They would all bind equally well • (e) None of them would bind at all. Biochem:Mechanisms,Regulation

38. Convergent evolution • Reappearance of ser-his-asp triad in unrelated settings • Subtilisin: externals very different from mammalian serine proteases; triad same Biochem:Mechanisms,Regulation

39. Subtilisin mutagenesis • Substitutions for any of the amino acids in the catalytic triad has disastrous effects on the catalytic activity, as measured by kcat. • Km affected only slightly, since the structure of the binding pocket is not altered very much by conservative mutations. • An interesting (and somewhat non-intuitive) result is that even these "broken" enzymes still catalyze the hydrolysis of some test substrates at much higher rates than buffer alone would provide. I would encourage you to think about why that might be true. Biochem:Mechanisms,Regulation

40. Cysteinyl proteases • Ancestrally related to ser proteases? • Cathepsins, caspases, papain • Contrasts: • Cys —SH is more basicthan ser —OH • Residue is less hydrophilic • S- is a weaker nucleophile than O- Diagram courtesy ofMariusz Jaskolski,U. Poznan Biochem:Mechanisms,Regulation

41. Papain active site Diagram courtesy Martin Harrison,Manchester University Biochem:Mechanisms,Regulation

42. Hen egg-white lysozyme • Antibacterial protectant ofgrowing chick embryo • Hydrolyzes bacterial cell-wall peptidoglycans • “hydrogen atom of structural biology” • Commercially available in pure form • Easy to crystallize and do structure work • Available in multiple crystal forms • Mechanism is surprisingly complex HEWLPDB 2vb10.65Å15 kDa Biochem:Mechanisms,Regulation

43. Mechanism of lysozyme • Strain-induced destabilization of substrate makes the substrate look more like the transition state • Long arguments about the nature of the intermediates • Accepted answer: covalent intermediate between D52 and glycosyl C1(Garrett & Grisham, fig. 14.39B) Biochem:Mechanisms,Regulation

44. The controversy Biochem:Mechanisms,Regulation

45. Regulation of enzymes • The very catalytic proficiency for which enzymes have evolved means that their activity must not be allowed to run amok • Activity is regulated in many ways: • Thermodynamics • Enzyme availability • Allostery • Post-translational modification • Protein-protein interactions Biochem:Mechanisms,Regulation

46. Thermodynamics as a regulatory force • Remember that Go (or Go’) is not the determiner of spontaneity: G is. • Therefore: local product and substrate concentrations determine whether the enzyme is catalyzing reversible reactions to the left or to the right • Rule of thumb: Go’ < -20 kJ mol-1 is irreversible Biochem:Mechanisms,Regulation

47. Enzyme availability • The enzyme has to be where the reactants are in order for it to act • Even a highly proficient enzyme has to have a nonzero concentration • How can the cell control [E]tot? • Transcription (and translation) • Protein processing (degradation) • Compartmentalization Biochem:Mechanisms,Regulation

48. Transcriptional control • mRNAs have short lifetimes • Therefore once a protein is degraded, it will be replaced and available only if new transcriptional activity for that protein occurs •  Many types of transcriptional effectors • Proteins can bind to their own gene • Small molecules can bind to gene • Promoters can be turned on or off Biochem:Mechanisms,Regulation

49. Protein degradation • All proteins havefinite half-lives; • Enzymes’ lifetimes often shorter than structural or transport proteins • Degraded by slings & arrows of outrageous fortune; or • Activity of the proteasome, a molecular machine that tags proteins for degradation and then accomplishes it Biochem:Mechanisms,Regulation

50. How the proteasome works • Proteins in need of degradation are tagged by covalent linkage to the small protein ubiquitin, or to a chain of several ubiquitin molecules • Proteasome is molecular machine that recognized ubiquitinated proteins • Cleaves off the ubiquitin(s) for re-use • Proteolytically degrades our initial protein Biochem:Mechanisms,Regulation