1. Unit 3 Enzymes. Catalysis and enzyme kinetics.

2. 3.1. Characteristics of biological catalysts. Coenzymes, cofactors, vitamins Enzyme nomenclature and classification 3.2. Enzyme catalysis. Transition state Active site Enzyme-substrate complex Factors involved in enzyme catalysis 3.3. Enzyme kinetics. Steady-state assumption and Michaelis-Menten equation Factors affecting the enzymatic activity Enzymatic inhibition Reversible inhibition Irreversible inhibition 3.4. Enzyme regulation. Allosteric behaviour Covalent modification Proteolysis OUTLINE

3. What characteristics features define enzymes? High catalytic power: ratio of the catalysed rate to the uncatalysed rate of the reaction = 10 6 -10 20 Enzymes are recover after each catalytic cycle. High specificity: (even stereospecifivity) Regulation The biological catalysts are: – Proteins (enzymes) – Catalytic RNA (ribozymes) 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

4. It converts 6x10 5 molecules per second 10 7 times faster than the uncatalysed reaction Ejemplos de reacciones catalizadas 10 11 times faster than the uncatalysed reaction The specificity depends on the R1 group. Protease Carbonic anhydrase 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

5. Nonprotein components required for the enzymatic activity: cofactor – Apoenzyme + cofactor = holoenzyme – Two types of cofactors: Metal ions: Mg 2+, Zn 2+, Cu 2+, Mn 2+,... Coenzymes: small organic molecules synthesised from vitamins. Prosthetic groups: tightly bound coenzymes Cofactors deficiency promotes some health problems. COFACTORS, COENZYMES AND VITAMINS 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

6. COFACTORS, COENZYMES AND VITAMINS

7. 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS COFACTORS, COENZYMES AND VITAMINS

8. NºClassReactionExamples 1OxidoreductasesOxidation-reduction reactionsGlucose oxidase (EC 1.1.3.4) 2TransferasesTransfer of functional groupsHexokinase (EC 2.7.1.2) 3HydrolasesHydrolysis reactionsCarboxipeptidase A (EC 3.4.17.1) 4LyasesAddition to double bondsPiruvate decarboxylase (EC 4.1.1.1) 5IsomerasesIsomerisation reactionsMalate isomerase (EC 5.2.1.1) 6LigasesFormation ob bonds (C-C, C-S, C- O and C-N) with ATP cleavage Piruvate carboxylase (EC 6.4.1.1) ENZYME NOMENCLATURE AND CLASSIFICATION 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

9. Traditional Nomenclature urease: urea hydrolysis amylase: starch hydrolysis DNA polymerase: Nucleotides polymerization Trivial designations (Ambiguity) Systematic Nomenclature (identify the substrate and the reaction) ATP + D-glucose  ADP + D-glucose 6-phosphate ATP: D-hexose 6-phosphotransferase hexokinase (traditional nomenclature) ENZYME NOMENCLATURE AND CLASSIFICATION 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

10. Carboxipeptidase A (peptidyl-L-amino acid hydrolase) EC 3.4.17.1 Class: 3  Hydrolases. Subclass: 4  peptide bond 17  metallocarboxypeptidases. Entry number: 1 A series of four number serves to specify a particular enzyme. The numbers are preceded by the letters EC (enzyme commission). First number: class Second number: subclass (electron donors, type of substrate, etc.) Third number: characteristics of the reaction (functional groups, etc.) Fourth number: order of the individual entries ENZYME NOMENCLATURE AND CLASSIFICATION 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

11. The conversion of S to P occurs because a fraction of the S molecules has the energy necessary to achieve a reactive condition known as the transition state (S-P intermediate) Enzymes (catalysts) work by lowering the free energy of activation related to the transition state A-B + C A …. B …. C A + B-C Ej. A-B + CA + B-C Transition state 3.2. ENZYME CATALYSIS

12. Substrate binds at the active site of the enzyme through relatively weak forces (chymotrypsin) Specificity Catalytic power Active site 3.2. ENZYME CATALYSIS

13. Lock and key theory (Fisher, 1890) Induced fit theory (Koshland y Neet, 1968) Enzyme-substrate complex interactions 3.2. ENZYME CATALYSIS

14. Glucose induced conformational change of hexokinase D-glucose (a)Unligaded form of hexoquinase and free glucose (b) Conformation of hexokinase with glucose bound Enzyme-substrate complex interactions 3.2. ENZYME CATALYSIS

15. FACTORS INVOLVED IN ENZYME CATALYSIS Proximity and orientation Surface phenomena Bounds tension Presence of reactive groups 3.2. ENZYME CATALYSIS

16. Proximity and orientation FACTORS INVOLVED IN ENZYME CATALYSIS 3.2. ENZYME CATALYSIS

17. FACTORS INVOLVED IN ENZYME CATALYSIS Bounds tension 3.2. ENZYME CATALYSIS

18. Mechanisms of catalysis General acid-base catalysis: proton transference in the transition state (from or towards the substrate) Covalent catalysis: transitory covalent bond between enzyme and substrate Metal ion catalysis: it acts as electrophilic catalysts, it promotes redox reactions, it stabilised charges, the polarity of certain bounds can change because of the metals… Presence of reactive groups FACTORS INVOLVED IN ENZYME CATALYSIS 3.2. ENZYME CATALYSIS

19. FACTORS INVOLVED IN ENZYME CATALYSIS 3.2. ENZYME CATALYSIS

20. General acid-base catalysis and covalent catalysis: protease Presence of reactive groups FACTORS INVOLVED IN ENZYME CATALYSIS 3.2. ENZYME CATALYSIS

21. Enolase General acid-base catalysis and metal ion catalysis FACTORS INVOLVED IN ENZYME CATALYSIS 3.2. ENZYME CATALYSIS

22. It is the analysis of the velocity (or rate) of a chemical reaction catalysed by an enzyme, and how the velocities can change on the basis of environmental parameters modifications. WHAT DO YOU HAVE TO KNOW? How the rate of an enzyme-catalysed reaction can be defined in a mathematical way Velocity units What is the order of a reaction (first-order reaction/second order reaction? 3.3. ENZYME KINETICS

23. Hypothetical enzyme catalyzing: S  P The rate of the reaction decreased when S is converted into P. Initial velocity: slope of tangent to the line at time 0 The rate of a enzymatic reactions depends on the substrate concentration 3.3. ENZYME KINETICS

24. The rate of a enzymatic reactions depends on the substrate concentration

25. Michaelis-Menten equation describes a curve known as a rectangular hyperbola The velocity of the product formation is: [ES] depends on: the velocity of ES formation from E + S the velocity of its dissociation to regenerate E+S or to form E + P. STEADY-STATE ASSUMPTION AND MICHAELIS-MENTEN EQUATION E + S  ES E + P k1k1 k -1 k2k2 3.3. ENZYME KINETICS

26. Concentration 0 Time Early stage ES formation Steady state [ES] is constant Steady-state Under experimental conditions [S]>>>[E]. The [ES] quickly reaches a constant value in such dynamic system, and remains constant until complete P formation: Steady State assumption 3.3. ENZYME KINETICS

27. K M, Michaelis constant Maximal velocity is obtained when the enzyme is saturated: [E] T =[ES] Michaelis-Menten Equation Steady-state 3.3. ENZYME KINETICS ][][]][[,0 ][ 211 ESk kSEk dt ESd   so

28. 3.3. ENZYME KINETICS

29. What does K M mean? When [S]=K M, v=V max /2 K M is the substrate concentration that gives a velocity equal to one—half the maximal velocity. Units of molarity. It indicates how efficient in an enzyme selecting substrates (specificity) Usually K M is used as a parameter to estimate the affinity of an enzyme for their substrates. K M is similar to the ES dissociation constant when k 2 <<k -1. E + S  ES E + P k1k1 k -1 k2k2 3.3. ENZYME KINETICS

30. The rate of a enzymatic reactions depends on the substrate concentration Michaelis-Menten

31. Turnover number, K cat K cat of an enzyme is a measure of its maximal catalytic activity. It represents the kinetic efficiency of the enzyme In the reactionk cat = k 2 K cat : turnover number: number of substrate molecules converted into product per enzyme molecule per unit time, when the enzyme is saturated with substrate First order velocity constant. Units: s -1 E + S  ES E + P k1k1 k -1 k2k2 3.3. ENZYME KINETICS

32. Turnover number, K cat

33. k cat /K M defines the catalytic efficiency of an enzyme It provides information about two combined facts: substrate binding and catalysis (substrate conversion into product). When [S]<<K M, K cat /K m is the velocity constant of the E +S conversion into E + P. Second order constant. Units: M -1 s - 1 The catalytic efficiency of an enzyme cannot exceed the diffusion-controlled rate of combination of E and S to form ES. 3.3. ENZYME KINETICS

34. Experimental determination of K M and V max Several rearrangements of the Michaelis-Menten equation transform it into a straight-line equation: Lineweaver-Burk double-reciprocal plot: 3.3. ENZYME KINETICS

35. Factors affecting the enzymatic activity Enzyme concentration -Enzymatic activity international unit (U): quantity of enzyme able to transform 1.0  mol substrate per minute at 25ºC (under optimal conditions) - Specific enzymatic activity (U/mg): number of enzymatic unit per mg of purified protein. It indicates how pure the enzyme is. Balls: they represent proteins Red balls: enzyme molecules Both cylinders: same activity units Right cylinder shows higher specific activity than the left cylinder 3.3. ENZYME KINETICS

36. Temperature The rates of enzyme-catalysed reactions generally increase with increasing temperature. However, at high temperatures the activity declines because of the thermal denaturation of the protein structure. pH Enzymes in general are active only over a limited pH range, and most have a particular pH at which their catalytic activity is optimal. pH changes can modify side chain, prosthetic groups and substrate charges, and consequently, the activity of the enzyme. Factors affecting the enzymatic activity 3.3. ENZYME KINETICS

37. Enzymatic inhibition Inhibition: velocity of an enzymatic reaction is decreased or inhibited by some agent (inhibitors) – Irreversible Inhibitor causes stable, covalent alterations in the enzyme – Examples: » Ampicillin: causes covalent modification of a transpeptidase catalysing the synthesis of the bacterial cellular wall » Aspirin: causes covalent modification in a cyclooxygenase involved in inflammation – Reversible Inhibitor interact with the enzyme through noncovalent association/dissociation reactions. 3.3. ENZYME KINETICS

38. REVERSIBLE INHIBITION  The inhibitor binds reversibly to the enzyme at the same site as substrate. The inhibitor resemble S structurally.  S-binding and I-binding are mutually exclusive, competitive processes.  The inhibition is blocked when the substrate concentration increases.  K mapp increases and V is unaffected Competitive Inhibition 3.3. ENZYME KINETICS

39. Competitive Inhibition REVERSIBLE INHIBITION 3.3. ENZYME KINETICS

40. Noncompetitive inhibition  Inhibitor interacts with both E and ES.  The inhibition is not blocked when the substrate concentration increases.  V app decreases and K m is unaffected REVERSIBLE INHIBITION 3.3. ENZYME KINETICS

41. Noncompetitive inhibition REVERSIBLE INHIBITION 3.3. ENZYME KINETICS

42.  Inhibitor only combines with ES  It does not bind in the active site.  V app and Km app decrease Uncompetitive inhibition REVERSIBLE INHIBITION 3.3. ENZYME KINETICS

43. Uncompetitive inhibition REVERSIBLE INHIBITION 3.3. ENZYME KINETICS

45. Chymotrypsin inhibition by diisopropylfluorophosphate (DIFP) Ciclooxigenase inhibition by aspirin IRREVERSIBLE INHIBITION 3.3. ENZYME KINETICS

46. Living systems must regulate the enzymatic catalytic activity to: - Coordinate metabolic processes - Promote adaptations to environmental changes - Growth and complete the living cycle in the correct way Two mechanisms of regulation: 1.- Control of the enzyme availability 2.- Control of the enzymatic activity, by means of modifications of the conformation or structure 3.4. ENZYME REGULATION

47. Allosteric enzyme: Oligomeric organization (more than one active site and more than one effector-binding site) The regulatory effects exerted on the enzyme’s activity are achieved by conformational changes occurring in the protein when effector metabolites bind Conformational states for a protein (monomer): Taut state (T): Low substrate affinity Relaxed state (R) : High substrate affinity ALLOSTERIC REGULATION 3.4. ENZYME REGULATION

48. Homotropic effect: The ligand- induced conformational change in one subunit can affect the adjoining subunit: Cooperativity Usually, it is positive regulation No Michaelis-Menten kinetics Sigmoidal curves ALLOSTERIC REGULATION 3.4. ENZYME REGULATION

49. Heterotropic effect: The effectors do not bind in the active site Activator: R state is stabilised Inhibitors: T state is stabilised ALLOSTERIC REGULATION 3.4. ENZYME REGULATION

50. Aspartate carbamoyltransferase: allosteric enzyme As product accumulates, the rate of the enzymatic reaction decreases (negative effect) Feedback inhibition 3.4. ENZYME REGULATION

51. Aspartate carbamoyltransferase: allosteric enzyme

52. COVALENT MODIFICATION 3.4. ENZYME REGULATION

53. Most of the covalent modification involved in enzyme activity regulation are phosphorylations. One or more than one phosphorylation site Protein kinases: They act in covalent modifications by attaching a phosphoryl moiety to target proteins Phosphoprotein phosphatases: They catalyse the removal of phosphate groups. COVALENT MODIFICATION 3.4. ENZYME REGULATION

54. Glucogen phosphorylase (adrenalina) COVALENT MODIFICATION 3.4. ENZYME REGULATION

55. Some proteins are synthesized as inactive precursors, called zymogens or proenzymes, that acquire full activity only upon specific proteolytic cleavage of one or several of their peptide bonds It is not energy dependent The peptide bond cleavage is irreversible Examples  Digestive enzymes  Blood clotting  Peptidic hormone (insulin)  Collagen  Caspases: apoptosis 3.4. ENZYME REGULATION PROTEOLYSIS

56. Trypsin cleaves the peptide bond joining Arg 15 - Ile 16 Chymotrypsin π is an enzymatically active form that acts upon other Chymotrypsin π molecules, excising two peptides. The end product is the mature protease Chymotrypsin α, in which the three peptide chains remain together because they are linked by two disulfide bonds PROTEOLYSIS COVALENT MODIFICATION 3.4. ENZYME REGULATION