Enzyme Mechanisms and Regulation

1. Enzyme Mechanisms and Regulation Andy HowardIntroductory Biochemistry, Fall 2008Tuesday 28 October 2008 Biochemistry: Mechanisms

2. How do enzymes reduce activation energies? • We can illustrate mechanistic principles by looking at specific examples; we can also recognize enyzme regulation when we see it. Biochemistry: Mechanisms

3. Regulation Thermodynamics Enzyme availability Allostery, revisited Mechanisms Induced-fit Tight Binding of Ionic Intermediates Serine proteases Other proteases Lysozyme Mechanism Topics Biochemistry: Mechanisms

4. 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. Biochemistry: Mechanisms

5. 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. Biochemistry: Mechanisms

6. 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. Biochemistry: Mechanisms

7. Proline racemase • Pyrrole-2-carboyxlate resembles planar transition state Biochemistry: Mechanisms

8. Yeast aldolase • Phosphoglycolohydroxamate binds much like the transition state to the catalytic Zn2+ Biochemistry: Mechanisms

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

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

11. 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 Biochemistry: Mechanisms

12. Example: 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 Biochemistry: Mechanisms

13. Hexokinase structure • Diagram courtesy E. Marcotte, UT Austin Biochemistry: Mechanisms

14. 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 Biochemistry: Mechanisms

15. 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 Biochemistry: Mechanisms

16. 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 aas at position 1, 2, -1, . . . • Others are more promiscuous CH NH C NH C NH R1 CH O R-1 Biochemistry: Mechanisms

17. Mechanism • Active-site serine —OH …Without neighboring amino acids, it’s fairly non-reactive • 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. Biochemistry: Mechanisms

18. 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. Biochemistry: Mechanisms

19. Diagram of first three steps Biochemistry: Mechanisms

20. Diagram of last four steps Diagrams courtesy University of Virginia Biochemistry: Mechanisms

21. 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 Biochemistry: Mechanisms

22. 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 - 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. Biochemistry: Mechanisms

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

24. 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 Biochemistry: Mechanisms

25. 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. Biochemistry: Mechanisms

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

27. 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 Biochemistry: Mechanisms

28. iClicker quiz! • 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 Biochemistry: Mechanisms

29. iClicker quiz, question 2:Why are proteases often synthesized as zymogens? • (a) Because the transcriptional machinery cannot function otherwise • (b) To prevent the enzyme from cleaving peptide bonds outside of its intended realm • (c) To exert control over the proteolytic reaction • (d) None of the above Biochemistry: Mechanisms

30. Question 3: 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 Biochemistry: Mechanisms

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

32. 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. Biochemistry: Mechanisms

33. 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 Biochemistry: Mechanisms

34. Papain active site Diagram courtesy Martin Harrison,Manchester University Biochemistry: Mechanisms

35. 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 (14.7) HEWLPDB 2vb10.65Å15 kDa Biochemistry: Mechanisms

36. 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 (14.39B) Biochemistry: Mechanisms

37. The controversy Biochemistry: Mechanisms

38. 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 Biochemistry: Mechanisms

39. Thermodynamics as a regulatory force • Remember that 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 Biochemistry: Mechanisms

40. 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 Biochemistry: Mechanisms

41. 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 Biochemistry: Mechanisms

42. 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 Biochemistry: Mechanisms

43. Compartmentalization • If the enzyme is in one compartment and the substrate in another, it won’t catalyze anything • Several mitochondrial catabolic enzyme act on substrates produced in the cytoplasm; these require elaborate transport mechanisms to move them in • Therefore, control of the transporters confers control over the enzymatic system Biochemistry: Mechanisms

44. Allostery • Remember we defined this as an effect on protein activity in which binding of a ligand to a protein induces a conformational change that modifies the protein’s activity • Ligand may be the same molecule as the substrate or it may be a different one • Ligand may bind to the same subunit or a different one • These effects happen to non-enzymatic proteins as well as enzymes Biochemistry: Mechanisms

45. Substrates as allosteric effectors (homotropic) • Standard example: binding of O2 to one subunit of tetrameric hemoglobin induces conformational change that facilitates binding of 2nd (& 3rd & 4th) O2’s • So the first oxygen is an allosteric effector of the activity in the other subunits • Effect can be inhibitory or accelerative Biochemistry: Mechanisms

46. Other allosteric effectors (heterotropic) • Covalent modification of an enzyme by phosphate or other PTM molecules can turn it on or off • Usually catabolic enzymes are stimulated by phosphorylation and anabolic enzymes are turned off, but not always • Phosphatases catalyze dephosphorylation; these have the opposite effects Biochemistry: Mechanisms

47. Cyclic AMP-dependent protein kinases • Enzymes phosphorylate proteins with S or T within sequence R(R/K)X(S*/T*) • Intrasteric control:regulatory subunit or domain has a sequence that looks like the target sequence; this binds and inactivates the kinase’s catalytic subunit • When regulatory subunits binds cAMP, it releases from the catalytic subunit so it can do its thing Biochemistry: Mechanisms

48. Kinetics of allosteric enzymes • Generally these don’t obey Michaelis-Menten kinetics • Homotropic positive effectors produce sigmoidal (S-shaped) kinetics curves rather than hyperbolae • This reflects the fact that the binding of the first substrate accelerates binding of second and later ones Biochemistry: Mechanisms

49. T  R State transitions • Many allosteric effectors influence the equilibrium between two conformations • One is typically more rigid and inactive, the other is more flexible and active • The rigid one is typically called the “tight” or “T” state; the flexible one is called the “relaxed” or “R” state • Allosteric effectors shift the equilibrium toward R or toward T Biochemistry: Mechanisms