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Enzyme Mechanisms and Regulation

Enzyme Mechanisms and Regulation. Andy Howard Introductory Biochemistry, Fall 2010 Wednesday 22 September 2010. How do enzymes reduce activation energies?. We want to understand what is really happening chemically when an enzyme does its job.

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Enzyme Mechanisms and Regulation

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  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

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