عید سعید قربان بر شما مبارک

1. عید سعید قربان بر شما مبارک یلدای زیبا هم همینطور!

2. In the Name of Allah ENZYMES: TUMS …Make Life on Earth Possible Abolfazl Golestani, PhD

3. An Important Question: • Why should we as medical students, study and learn about the ENZYMS? • For answer go to slide No. 55 Enzymes; by: Dr. Abolfazl Golestani, PhD

4. Chemical reaction Catalyst AB Product(s) Reactant(s) Catalyst A +B B + C • Catalysts • Increase the rate of a reaction • Are not consumed in the reaction • Can act repeatedly Heat Acid Base Metals What are some of the known catalysts? Enzymes; by: Dr. Abolfazl Golestani, PhD

5. Enzyme is either a pure protein or may require a non-protein portion Apoenzyme = protein portion Apoenzyme + non-protein part = Holoenzyme According to Holum, the non-protein portion may be: • A coenzyme - a non-protein organic substance which is loosely attached to the protein part • A prosthetic group - an organic substance which is firmly attached to the protein or apoenzyme portion • A cofactor - these include K+, Fe++, Fe+++, Cu++, Co++, Zn++, Mn++, Mg++, Ca++, and Mo+++ Enzymes; by: Dr. Abolfazl Golestani, PhD

6. Basic enzyme reactions S + E  E + P S = Substrate P = Product E = Enzyme Swedish chemist Savante Arrhenius in 1888 proposed: Substrate and enzyme form some intermediate known as theEnzyme-Substrate Complex (ES): S + E  ES ES  P + E Binding step Catalytic step Enzymes; by: Dr. Abolfazl Golestani, PhD

7. There are two models of enzyme substrate interaction 1. Lock and key model; Emil Fischer (1890) • The active site: • Substrate Binding Site • Catalytic Site 2. Induced fit model; Daniel Koshland (1958) Enzymes; by: Dr. Abolfazl Golestani, PhD

8. Induced fit in Carboxypeptidase A Three amino acids are located near the active site (Arg 145, Tyr 248, and Glu 270) Enzymes; by: Dr. Abolfazl Golestani, PhD

9. S E Enzyme-Substrate complex is transient S + E P + E When the enzyme unites with the substrate, in most cases the forces that hold the enzyme and substrate are non-covalent. Binding forces of substrate are: • Ionic interactions: (+)•••••(-) • Hydrophobic interactions: (h)•••••(h) • H-bonds: O-H ••••• O, N-H ••••• O, etc. • van der Waals interactions Enzymes; by: Dr. Abolfazl Golestani, PhD

10. Some important characteristics of enzymes -Potent (high catalytic power) High reaction rates They increase the rate of reaction by a factor of 103-1012 -Efficient (high efficiency) catalytic efficiency is represented by Turnover number: moles of substrate converted to product per second per mole of the active site of the enzyme -Milder reaction conditions Enzymatically catalyzed reactions occur at mild temperature, pressure, and nearly neutral pH (i.e. physiological conditions) Enzymes; by: Dr. Abolfazl Golestani, PhD

11. Some important characteristics of enzymes, cont. -Specific (specificity) Substrate specific Reaction Specific Stereospecific -Capacity for regulation Enzymes can be activated or inhibited so that the rate of product formation responds to the needs of the cell -Location within the cell Many enzymes are located in specific organelles within the cell. Such compartmentalization serves: to isolate the reaction substrate from competing reactions, to provide a favorable environment for the reaction, and to organize the thousands of enzymes present in the cell into purposeful pathways. Enzymes; by: Dr. Abolfazl Golestani, PhD

12. Specificity Substrate Specificity • Absolute specificity: For example Urease • Functional Groupspecificity: For example OH, CHO, NH2. • Linkage specificity: For example Peptide bond. Reaction specificity • Yields are nearly 100% • Lack of production of by-products • Save energy and prevents waste of metabolites Stereospecificity • Enzymes can distinguish between enantiomers and isomers Enzymes; by: Dr. Abolfazl Golestani, PhD

13. Enzymes requiring metal ions as cofactors Enzymes; by: Dr. Abolfazl Golestani, PhD

14. Many vitamins are coenzyme precursors Enzymes; by: Dr. Abolfazl Golestani, PhD

15. Methods for naming enzymes (nomenclature) • Very old method: Pepsin, Renin, Trypsin • Old method: Protease,Lipase,Urease • Systematic naming (EC = Enzyme Commission number): • The name has two parts: • The first part: name of substrate (s) • The second part: ending in –ase, indicates the type of reaction. Additional information can follow in parentheses: • L-malate:NAD+ oxidoreductase (decarboxylating) Enzymes; by: Dr. Abolfazl Golestani, PhD

16. Each enzyme has anEC number = Enzyme Commission number • EC number consists of 4 integers: • The 1st designates to which of the six major classes an enzyme belongs • The 2nd integer indicates a sub class, e.g. type of bond • The 3rdnumber is a subclassification of the bond type or the group transferred in the reaction or both (a subsubclass) • The 4th number is simply a serial number Enzymes; by: Dr. Abolfazl Golestani, PhD

17. There are six functional classes of enzymes Enzymes; by: Dr. Abolfazl Golestani, PhD

18. Enzymes; by: Dr. Abolfazl Golestani, PhD

19. EC Classification Class Subclass Sub-subclass Serial number Enzyme Nomenclature and Classification Enzymes; by: Dr. Abolfazl Golestani, PhD

20. Example of Enzyme Nomenclature • Common name(s): Invertase, sucrase • Systematic name: -D-fructofuranoside fructohydrolase (E.C. 3.2.1.26) • Recommended name: -fructofuranosidase Enzymes; by: Dr. Abolfazl Golestani, PhD

21. Kinetic Enzyme kinetics Enzymes; by: Dr. Abolfazl Golestani, PhD

22. Energy barrier = Free Energy of Activation X T* Y T=Transition state (Ea) Thermodynamics: Type (Exergonic or Endergonic) Kinetics: How fast the reaction will proceed Enzymes; by: Dr. Abolfazl Golestani, PhD

23. Enzyme Stabilizes Transition State What’s the difference? Many enzymes function by lowering the activation energy of reactions. Enzymes; by: Dr. Abolfazl Golestani, PhD Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166

24. عید سعید و عظیم غدیر بر شما مبارک ميلاد حضرت امام هادي (ع) را تهنيت مي گويم، میلاد حضرت مسیح (ع) را هم همین طور!

25. EA = Activation energy; a barrier to the reaction Can be overcome by adding energy....... ......or by catalysis Enzymes; by: Dr. Abolfazl Golestani, PhD

26. Enzymes Are Complementary to Transition State X If enzyme just binds substrate then there will be no further reaction Enzyme not only recognizes substrate, but also induces the formation of transition state, see also Enz01 Enzymes; by: Dr. Abolfazl Golestani, PhD

27. Active Site Is a Deep Buried Pocket Why energy required to reach transition state is lower in the active site? It is a magic pocket (1) Stabilizes transition + (2) Expels water CoE (2) (1) (3) Reactive groups (4) - (4) Coenzyme helps (3) Enzymes; by: Dr. Abolfazl Golestani, PhD Juang RH (2004) BCbasics

28. Active Site Avoids the Influence of Water + - Preventing the influence of water sustains the formation of stable ionic bonds Enzymes; by: Dr. Abolfazl Golestani, PhD Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.115

29. Enzyme Reaction Mechanism • Consider for example the mechanism of Chymotrypsin: • Enz06 • Enz07 Enzymes; by: Dr. Abolfazl Golestani, PhD

30. Modes of rate enhancement • Facilitation of Proximity • Increase the Effective concentration • Hold reactants near each other in proper orientation • Strain, Molecular Distortion, and Shape Change • Put a strain on susceptible bonds • General Acid –Base Catalysis • Transfer of a proton in the transition state • Covalent Catalysis • Form covalent bond with substrate destabilization of the substrate Enzymes; by: Dr. Abolfazl Golestani, PhD

31. Factors Affecting Rate of Enzyme Reactions • Temperature • pH • Enzyme concentration [E] • Substrate concentration [S] • Inhibition • Regulation (Effectors) Enzymes; by: Dr. Abolfazl Golestani, PhD

32. 1- Optimum Temperature • Little activity at low temperature (low number of collisions) • Rate increases with temperature (more successful collisions); rate doubles for every 10°C increase in temperature • Most active at optimum temperatures (usually 37 oC in humans) • Enzymes isolated from thermophilic organisms display maxima around 100 °C • Enzymes isolated from psychrophilic organisms display maxima around 10 °C. • Activity lost with denaturation at high temperatures Enzymes; by: Dr. Abolfazl Golestani, PhD

33. 2- Optimum pH • Effect of pH on ionization of active site • Effect of pH on enzyme denaturation • Each enzyme has an optimal pH (~ 6 - 8 ) • Exceptions : digestive enzymes in the stomach (pH 2) digestive enzymes in the intestine (pH 8) Enzymes; by: Dr. Abolfazl Golestani, PhD

34. 3- Enzyme concentration • The Rate (v) of reaction Increases proportional to the enzyme concentration [E] ([S] is high) Enzymes; by: Dr. Abolfazl Golestani, PhD

35. 4- Substrate concentration • When enzyme concentration is constant, increasing [S] increases the rate of reaction, BUT • Maximum activity reaches when all E combines with S (when all the enzyme is in the ES form) Enzymes; by: Dr. Abolfazl Golestani, PhD

36. 0 1 2 3 4 5 6 7 8 80 60 40 20 0 Product (v) 0 2 4 6 8 Substrate (mmole) [S] Enzyme Velocity Curve, see also Enz02 S + E P (in a fixed period of time) Constant [E] Enzymes; by: Dr. Abolfazl Golestani, PhD Juang RH (2004) BCbasics

37. بسم الله الرحمن الرحیم Enzymes 3rd part Enzymes; by: Dr. Abolfazl Golestani, PhD

38. K+1 k2 S E k-1 maximal velocity, Vmax 0.5Vmax Km Michaelis-Menten Equation S E P Enzymes; by: Dr. Abolfazl Golestani, PhD

39. MM Equation Derivation (steady state) Enzymes; by: Dr. Abolfazl Golestani, PhD

40. Practical Summary - Vmax and Km • Vmax • How fast the reaction can occur under ideal circumstances • Km • Range of [S] at which a reaction will occur • Binding affinity of enzyme for substrate • LARGER Km  the WEAKER the binding affinity Enzymes; by: Dr. Abolfazl Golestani, PhD

41. Practical Summary, cont. Enzymes; by: Dr. Abolfazl Golestani, PhD

42. Practical Summary; cont. • Kcat/Km • Practical idea of the catalytic efficiency, i.e. how often a molecule of substrate that is bound reacts to give product Enzymes; by: Dr. Abolfazl Golestani, PhD

43. Order of Reaction • When [S] << Km vo = (Vmax/Km )[S] 2. When [S] = Km vo = Vmax/2 3. When [S] >> Km vo = Vmax zero order Mixed order 2 First order Enzymes; by: Dr. Abolfazl Golestani, PhD

44. k1 k2 S E k-1 Importance of Viin Measurement of Enzyme Activity S E P • Working with vo minimizes complications with • reverse reactions • product Inhibition The rate of the reaction catalyzed by an enzyme in a sample is expressed in Units. Units = V = activity = Micromoles (mol; 10-6 mol or ….), of substrate reacting or product produced per min. It is better to measure it at linear part of the curve Enzymes; by: Dr. Abolfazl Golestani, PhD

45. 1 vo vo 1 Vmax - 1 Km 1/S S Lineweaver-Burk plot 1/2 Km Direct plot Double reciprocal plot Juang RH (2004) BCbasics Enzymes; by: Dr. Abolfazl Golestani, PhD

46. Allosteric Enzymes • Why the sigmoid shape? • Allosteric enzymes are multi-subunit enzymes, each with an active site • They show a cooperative response to substrates • See Enz13 hyperbolic curve; Michaelis-Menten kinetics Sigmoidal curve Enzymes; by: Dr. Abolfazl Golestani, PhD

47. IrreversibleInhibition=Enzyme Stops Working Permanently • Destruction of enzyme • Irreversible Inhibitor=forms covalent bonds to E (inactive E) Examples: • Diisopropylfluorophosphate • inhibits acetylcholine esterase • binds irreversibly to –OH of serine residue • Cyanide and sulfide • Inhibit cytochrome oxidase • bind to the iron atom • Fluorouracil • inhibits thymidine synthase (suicide inhibition - metabolic product is toxic ) • Aspirin • Inhibits prostaglandin synthase • acylates an amino group of the cyclooxygenase Enzymes; by: Dr. Abolfazl Golestani, PhD

48. Reversible Inhibition=Temporary Decrease of Enzyme Function • Three types based on “how increasing [S] affects degree of inhibition”: • Competitive: degree of inhibition decreases • Non-competitive: degree of inhibition is unaffected • Anti- or Uncompetitive: degree of inhibition increases • The Lineweaver-Burk plot is useful in determining the mechanisms of actions of various inhibitors, see Enz04 Enzymes; by: Dr. Abolfazl Golestani, PhD

49. The Effects of Enzyme Inhibitors Enzymes; by: Dr. Abolfazl Golestani, PhD

50. Example • When a slice of apple is exposed to air, it quickly turns brown. This is because the enzyme o-diphenyl oxidase catalyzes the oxidation of phenols in the apple to dark-colored products. • Catechol can be used as the substrate. The enzyme converts it into o-quinone(A), which is then further oxidized to dark products. Enzymes; by: Dr. Abolfazl Golestani, PhD