Enzymes V: Specific Mechanisms; Regulation

1. Enzymes V:Specific Mechanisms; Regulation Andy HowardIntroductory Biochemistry 10 November 2008 Biochem: Specific Mechanisms

2. Examples of mechanisms • We’ll look at the serine protease mechanism in detail, and then explore a few other mechanisms to illustrate specific ideas • Then we’ll begin our discussion of regulation of enzymes Biochem: Specific Mechanisms

3. Serine Proteases Significance Catalytic residues Sequence of events Chymotrypsin Evolution Other mechanisms Cysteinyl proteases Lysozyme TIM Regulation by thermodynamics Enzyme availability Transcription Degradation Compartmentation Allostery Mechanisms and Regulation Biochem: Specific Mechanisms

4. 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: Specific Mechanisms

5. 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 Biochem: Specific Mechanisms

6. Mechanism • Active-site serine —OH …Without neighboring amino acids, it’s fairly non-reactive (naked ser-OH pKa ~ 14) • 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: Specific Mechanisms

7. 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: Specific Mechanisms

8. Diagram of first three steps Biochem: Specific Mechanisms

9. Diagram of last four steps Diagrams courtesy University of Virginia Biochem: Specific Mechanisms

10. 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: Specific Mechanisms

11. 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: Specific Mechanisms

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

13. 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: Specific Mechanisms

14. What mechanistic concepts do serine proteases not illustrate? • Quaternary structural effects(We’ll discuss this under regulation…) • Protein-protein interactions(Becoming increasingly important) • Allostery(also will be discussed under regulation) • Noncompetitive inhibition Biochem: Specific Mechanisms

15. 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: Specific Mechanisms

16. 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: Specific Mechanisms

17. 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 Biochem: Specific Mechanisms

18. Non-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: Specific Mechanisms

19. 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 Biochem: Specific Mechanisms

20. Question 3: what would bind tightest in the TIM active site? • (a) DHAP (substrate) • (b) D-glyceraldehyde-3-P (product) • (c) 2-phosphoglycolate(Transition-state analog) • (d) They would all bind equally well Biochem: Specific Mechanisms

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

22. 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: Specific Mechanisms

23. 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: Specific Mechanisms

24. Papain active site Diagram courtesy Martin Harrison,Manchester University Biochem: Specific Mechanisms

25. 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 Biochem: Specific Mechanisms

26. 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) Biochem: Specific Mechanisms

27. The controversy Biochem: Specific Mechanisms

28. Triosephosphate isomerase(TIM) • dihydroxyacetone phosphate  glyceraldehyde-3-phosphate Glyc-3-P  DHAP Km=10µM kcat=4000s-1 kcat/Km=4*108M-1s-1 Biochem: Specific Mechanisms

29. TIM mechanism • DHAP carbonyl H-bonds to neutral imidazole of his-95; proton moves from C1 to carboxylate of glu165 • Enediolate intermediate (C—O- on C2) • Imidazolate (negative!) form of his95 interacts with C1—O-H) • glu165 donates proton back to C2 • See Fort’s treatment (http://chemistry.umeche.maine.edu/CHY431/Enzyme3.html) Biochem: Specific Mechanisms

30. Enzymes are under several levels of control • Some controls operate at the level of enzyme availability • Other controls are exerted by thermodynamics, inhibition, or allostery Biochem: Specific Mechanisms

31. 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: Specific Mechanisms

32. 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 Biochem: Specific Mechanisms

33. 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: Specific Mechanisms

34. 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: Specific Mechanisms

35. 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: Specific Mechanisms

36. 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 Biochem: Specific Mechanisms

37. 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 Biochem: Specific Mechanisms

38. 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 Biochem: Specific Mechanisms

39. 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 Biochem: Specific Mechanisms