2. Enzymes O -CO -C Hi, Everybody!

3. Summarize the various methods of enzyme regulation. Intended learning outcomes(ILO)

4. Objectives Enzymes as Biological Catalysts The Properties of Enzymes Enzyme classification Enzyme kinetics Factors affecting enzyme activity Enzyme Inhibition Regulation of enzyme activity. Applications of Enzyme Action

5. Enzyme regulation

6. Regulatory enzymes A A B C C D D E1 E2 E3 KEY ENZYME Rate limiting enzyme REGULATORY ENZYME (Catalyze the first step unique to particular pathway KEY ENZYME Rate limiting enzyme REGULATORY ENZYME (Catalyze the first step unique to particular pathway Rate limited step Committed step (conversion of B to C) Usually irreversible Rate limited step Committed step (conversion of B to C) Usually irreversible Regulatory enzyme adjust the overall rate of the pathway to meet the cell‘s demand

7. Control of the metabolic pathway Key enzymes Regulatory enzymes Rate limiting enzymes

8. Enzyme regulation ENZYME QUANTITY (long term) ENZYME CATALYTIC ACTIVITY(short term)

9. Regulation of enzyme quantity The absolute amount of the enzyme is the number of enzyme molecules available. It is determined by the rate of enzyme turn over Slow in the human, occurring over hours to days

10. Regulation of enzyme quantity Enzyme quantity Enzyme synthesis(Long term) at gene level InductionRepression Enzyme degradation

11. 1-Regulation of enzyme synthesis

12. Stimulation of Enzyme Synthesis in response to an inducer Inducer Substrate Glukokinase stimulated by glucose Hormone Insulin induce the synthesis of glycokinase enzyme after carbohydrate meal A- Induction

13. protein transcription cytoplasm nucleus translation

14. Constitutive enzymes A non inducible enzymes = Constitutive enzymes A non inducible enzymes = Constitutive enzymes

15. Inhibition of Enzyme Synthesis in response to an repressor Repressor Hormone Glucagon repress glukokinase during starvation Product of the enzyme heme repress ALA symnthase (heme synthesis) B- Repression

16. 1-Regulation of enzyme degradation Absence of dietary factors, substrate, coenzymes or metal ions Alter the enzyme conformation Enzyme more susceptible to proteolysis

17. Regulation of catalytic activities (short term regulation)

18. Mechanisms of Regulation of catalytic activities 1.ALLOSTERIC REGULATION 2. REVERSIBLE COVALENT MODIFICATION 3.Irreversible covalent modification 4. protein-protein interaction between regulatory and catalytic subunits 5-Compartmention 6-Multienzyme complex

19. 1-Allosteric Regulation (Allos= another site) Allosteric Enzyme Active site bind to substrate Allosteric site bind to allosteric modifier

20. Allosteric Regulation Allosteric enzymes have 2 binding sites: 1-Active site binds substrate 2-Allosteric site binds regulator (Effectors or modifiers)

21. Allosteric Regulation Binding of regulatory molecule to allosteric site. It induce a conformational change in the enzyme Increase the enzyme acivity(positive allosteric modifier ) Inhibit the enzyme acivity(negative allosteric modifier

23. Allosteric Regulation Binding of allosteric inhibitor is non covalent(reversible). Homotropic enzyme When the substrate itself acts as positive effector, homotropic effector Heterotropic enzyme When the effector is different from substrate acting as negative effector, heterotropic effector

24. K0.5 instead of Km Sigmoidal curve not hyperbolic curve. Michelis-Menten kinetics do not apply to allosteric enzymes Most allosteric enzymes are polymeric(two or more subunits). Allosteric Regulation

25. Allosteric effectors Some allosteric effectors change K0.5 but not Vmax They give double reciprocal plot like those of competitive inhibitors Some allosteric effectors change Vmax but not Km They give double reciprocal plot like those of non competitive inhibitors They alter the rate of breakdown of ES complex to products Few allosteric effectors alter both K0.5 and Vmax

26. Binding of the substrate at one active site Facilitates binding of other substrate molecules at other active sites on other subunits The enzyme shifts from taut(T)form to relaxed (R) form as the substrate is bound(O2 binding to hemoglobin) Sigmoid kinetics = cooperative binding of the substrate to the protein subunits of the enzyme

27. Positive allosteric modifiers Shift the enzyme to R form Enzyme binds to the substrate more easily Hyperbolic curve Negative allosteric modifiers Shift the enzyme to T form Enzyme binds to the substrate less readily Sigmoidal curve

28. Sigmoidal Curve Effect Sigmoidal curve Exaggeration of sigmoidal curve yields a drastic zigzag line that shows the On/Off point clearly Positive effector (ATP) brings sigmoidal curve back to hyperbolic Negative effector (CTP) keeps Consequently, Allosteric enzyme can sense the concentration of the environment and adjust its activity Noncooperative (Hyperbolic) Cooperative (Sigmoidal) CTP ATP vovo vovo [Substrate] OffOn Juang RH (2004) BCbasics

29. Mechanism and Example of Allosteric Effect A I [S] vovo vovo (+) (-) X X X R = Relax (active) T = Tense (inactive) Activation Inhibition KineticsCooperationModels (-) (+) Juang RH (2004) BCbasics

30. Feed- Back Inhibition A A B C C D D E1 E2 E3 Accumulation of end products Heterotropic Allosteric inhibition of first enzyme

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32. 2- Reversible Covalent modification Addition of a group to the enzyme protein by covalent bond Removal of a group by cleaving the covalent bond.

33. Reversible Covalent modification

34. 1-Phosphorylation/ Dephosphorylation (Most commonly used) 2-Methylation/ demethylation 3Acetylation/ deacetylation 4-Uridylation /deuridylation 5-ADP ribosylation 2-Reversible Covalent modification

35. Phosphorylation/Dephosphorylation Phosphorylation catalyzed by kinase Dephosphorylation catalyzed by phosphatase They increase or inhibit enzyme activity Require ATP OH of serine,thronine Under hormonal and neural control

36. phosphorylation Activate some enzyme Glycogen phosphorylase Inhibit other enzymes Glycogen synthase

37. Phosphorylation Conformational Change dephosphorylastion Phosphatase P Protein OH Ser Ser Thr Tyr (His) Active Inactive Inactive Active Glycogen phosphorylase bGlycogen phosphorylase a Kinase phosphorylation

38. 3-irreversible covalent modification (proteolytic cleavage)  Cleavage of small peptide  Reveal the catalytic site (active enzyme) (inactive zymogen)

39. 3-Proenzyme Irreversible form of covalent modification, Only reversed by specific inhibitors

40. 3-Proenzyme examples

41. Trypsin Pancreatic Trypsinogen  Cleavage of small peptide in the intestine Alpha one antitrypsin

42. 3-Proenzyme examples Thrombin Prothrombin  Cleavage of small peptide Antithrombin III

43. 4-Regulatory proteins are allosteric effectors that can either activate or inhibit enzyme to which they bind

44. cAMP Controls Protein Kinase A Activity R C R C R R A A A A A A A A C C Regulatory subunits Catalytic subunits cAMP Active kinase C CREB P Nucleus Activation Gene expression DNA

45. 5-Compartmentation Compartmentaion facilitates the regulation of any pathway independent of other pathways going on in other compartments of the cell e.g. enzymes of citric acid are all in the mitochondria. Localization of enzymes of particular pathway in one cell compartment control the absolute quantity of the enzymes

46. 5-Compartmentation A-opposing pathways(anabolic and catabolic) B-pathway may be partitioned away from its substrate) C-Shuttle mechanism D- Enclosing the hydrolytic enzymes in the lysosomes protect the cell against lysis

47. A-opposing pathways(anabolic and catabolic) are present in different compartments Enzymes involved in fatty acid synthesis Cytosol Enzymes involved in fatty acid oxidation Mitochondria

48. B-pathway may be partitioned away from its substrate Acetyl COA should be transported from mitochondria to enter the cytoplasm for fatty acid synthesis Cytosol fatty acids should enter mitochondria to be oxidized Mitochondria

49. C-SHUTTELE Mechanisms Solves the problem of translocation of metabolites across membranes by converting the metabolites into form that can penetrate barrier

50. 6-Multienzyme complex A single enzyme with multiple catalytic (enzymatic) activities Multiple enzymes are organized so that the product of one reaction become the substrate for the next adjacent enzymes without problem of compartments Coordinate control of enzyme complex Conformational change in one compartment is transmitted by protein-protein interaction to other components of the complex amplifying regulatory effect

53. 6-Multienzyme complex Fatty acid synthase enzyme complex is a dimer of two isentical polypeptides, each containing 7 enzymes required for fatty acid synthesis Inhibit accumulation of free intermediates Allow coordinate control of the enzyme complex

54. Enzyme Regulation Enzyme Amount Synthesis InductionRepression Degradation Enzyme ActivityCovalent Catalysis Reversible Irreversible (proenzyme ) Non Covalent Catalysis Allosteric Modulator proteins compartmentaion Multienzyme complex