Enzyme Regulation

1. Enzyme Regulation Andy HowardIntroductory Biochemistry 19 November 2014 Mechanisms & Regulation

2. Enzyme Regulation • Regulation happens at several levels • Even though it isn’t really an enzyme, hemoglobin can teach us how allostery in enzymes works. • But first, we’ll finish our discussion of mechanisms Mechanisms & Regulation

3. Globins Oxygen binding Structure R and T states Allostery Bohr effect BPG as an effector Sickle-cell anemia Mechanisms Cysteinyl proteases Lysozyme TIM Regulation Thermodynamics Availability Allostery PTM Protein-protein interactions Mechanisms & Regulation Mechanisms & Regulation

4. Cysteinyl proteases • Ancestrally related toserine proteases? • Cathepsins,caspases, papain • Contrasts: • Cys —SH is more basicthan ser —OH • Residue is less hydrophilic • S- is a weaker nucleophile than O- Carica papaya papain 24 kDa monomerEC 3.4.22.2PDB 1PPN, 1.6Å Mechanisms & Regulation

5. Papain active site Diagram courtesy Martin Harrison,Manchester University Mechanisms & Regulation

6. 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 Mechanisms & Regulation

7. 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 Mechanisms & Regulation

8. The controversy Mechanisms & Regulation

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

10. 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) Mechanisms & Regulation

11. 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 Mechanisms & Regulation

12. 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 Mechanisms & Regulation

13. 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 Mechanisms & Regulation

14. Why -20 kJ/mol? • ΔG = ΔGo’+ RT ln[Products]/[Reactants] • We ask: if ΔGo’ = -20 kJ/mol, what ratio of concentrations will make ΔG = 0? • That’s easy to answer:0 = -20 + RTln[products]/[reactants] • For T=300.66K, RT=2.5kJ/mol, that’s20 = 2.5ln[products]/[reactants] • ln[products]/[reactants] = 8,[products]/[reactants]=e8 = 2981… • How likely is that? Mechanisms & Regulation

15. 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 Mechanisms & Regulation

16. 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 Mechanisms & Regulation

17. 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 Mechanisms & Regulation

18. 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 Mechanisms & Regulation

19. 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 Mechanisms & Regulation

20. 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 Mechanisms & Regulation

21. 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 Mechanisms & Regulation

22. Cyclic AMP-dependent protein kinases • Enzymes phosphorylate proteins with S or T within sequenceR(R/K)X(S*/T*) • Intrasteric control:regulatory subunit or domain has sequence that resembles target sequence; this binds, inactivates the kinase’s catalytic subunit • When regulatory subunits bind cAMP, it releases from catalytic subunit so it can perform Mouse cAMP-dependent protein kinase EC 2.7.11.11174 kDa dimer of dimersPDB 3TNP, 2.3Å Mechanisms & Regulation

23. 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 Mechanisms & Regulation

24. 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 Mechanisms & Regulation

25. MWC model for allostery • Emphasizes symmetry and symmetry-breaking in seeing how subunit interactions give rise to allostery • Can only explain positive cooperativity Mechanisms & Regulation

26. Koshland (KNF) model • Emphasizes conformational changes from one state to another, induced by binding of effector • Ligand binding and conformational transitions are distinct steps • … so this is a sequential model for allosteric transitions • Allows for negative cooperativity as well as positive cooperativity Mechanisms & Regulation

27. Oligomerization and allostery • Often the RT transition is influenced by enzyme oligomerization • Tryptophan synthase: dimerization shifts equilibrium toward R • Amino acid kinases: hinge motions of monomers engage cooperativity Thermatoga N-acetylglutamate kinase: 194 kDa hexamer, trimer shownEC 2.7.2.8; PDB 2BTY, 2.75¸Å Mechanisms & Regulation

28. Heterotropic effectors Mechanisms & Regulation

29. Post-translational modification • We’ve already looked at phosphorylation • Proteolytic cleavage of the enzyme to activate it is another common PTM mode • Some proteases cleave themselves (auto-catalysis); in other cases there’s an external protease involved • Blood-clotting cascade involves a series of catalytic activations Mechanisms & Regulation

30. Zymogens • As mentioned earlier, this is a term for an inactive form of a protein produced at the ribosome • Proteolytic post-translational processing required for the zymogen to be converted to its active form • Cleavage may happen intracellularly, during secretion, or extracellularly Bacillus subtilisin + prosequence 35 kDa heterodimer EC 3.4.21.62 PDB 3CNQ, 1.71Å Mechanisms & Regulation

31. Blood clotting • Seven serine proteases in cascade • Final one (thrombin) converts fibrinogen to fibrin, which can aggregate to form an insoluble mat to prevent leakage • Two different pathways: • Intrinsic: blood sees injury directly • Extrinsic: injured tissues releasefactors that stimulate process • Come together at factor X Human thrombinEC 3.4.21.5 36kDa monomerPDB 3RM2, 1.23Å Mechanisms & Regulation

32. Cascade Human Factor XaEC 3.4.21.634kDa monomerPDB 2JKH, 1.25Å Mechanisms & Regulation

33. Protein-protein interactions • One major change in biochemistryin the last 25 years is the increasingemphasis on protein-protein interactions in understanding biological activities • Many proteins depend on exogenous partners for modulating their activity up or down • Example: cholera toxin’s enzymatic component depends on interaction with human protein ARF6 Vibrio cholerae toxin A1 subunit + human ARF6: 37 kDa heterodimerPDB 2A5D, 1.8Å Mechanisms & Regulation

34. 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 Mechanisms & Regulation

35. Globins as aids to understanding • Myoglobin and hemoglobin are well-understood non-enzymatic proteins whose properties help us understand enzyme regulation • Hemoglobin is described as an “honorary enzyme” in that it “catalyzes” the reactionO2(lung)  O2 (peripheral tissues) Mechanisms & Regulation

36. Setting the stage for this story • Myoglobin is a 16kDa monomeric O2-storage protein found in peripheral tissues • Has Fe-containing prosthetic group called heme; iron must be in Fe2+ state to bind O2 • It yields up dioxygen to various oxygen-requiring processes, particularly oxidative phosphorylation in mitochondria in rapidly metabolizing tissues Mechanisms & Regulation

37. Why is myoglobin needed? • Free heme will bind O2 nicely;why not just rely on that? • Protein has 3 functions: • Immobilizes the heme group • Discourages oxidation of Fe2+ to Fe3+ • Provides a pocket that oxygen can fit into Mechanisms & Regulation

38. Setting the stage II • Hemoglobin (in vertebrates, at least) is a tetrameric, 64 kDa transport protein that carries oxygen from the lungs to peripheral tissues • It also transports acidic CO2 the opposite direction • Its allosteric properties are what we’ll discuss Mechanisms & Regulation

39. Structure determinations Photo courtesyEMBL • Myoglobin & hemoglobin were the first 2 proteins to have their 3-D structures determined experimentally • Myoglobin: Kendrew, 1958 • Hemoglobin: Perutz, 1958 • Most of the experimental tools that crystallographers rely on were developed for these structure determinations • Nobel prizes for both, 1965(small T!) Photo courtesyOregon State Library Mechanisms & Regulation

40. Myoglobin structure Sperm whale myoglobin; 1.4 Å18 kDa monomerPDB 2JHO • Almost entirely -helical • 8 helices, 7-26 residues each • Bends between helices generally short • Heme (ferroprotoporphyrin IX) tightly but noncovalently bound in cleft between helices E&F • Hexacoordinate iron is coordinated by 4 N atoms in protoporphyrin system and by a histidine side-chain N (his F8): fig.15.25 • Sixth coordination site is occupied by O2, H2O, CO, or whatever else fits into the ligand site Mechanisms & Regulation