Biological catalysts

1. Enzymes TUMS Biological catalysts Azin Nowrouzi, PhD Tehran University of Medical Sciences

2. Chemical reaction Catalyst AB Product(s) Reactant(s) Catalyst A +B B + C • Catalysts • Increase the rate of a reaction. • Are not consumed by the reaction. • Can act repeatedly. Heat Acid Base Metals What are some of the known catalysts?

3. 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+++.

4. 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 substance known as TheEnzyme-Substrate Complex (ES): S + E  ES ES  P + E Binding step Catalytic step

5. 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)

6. Induced fit in Carboxypeptidase A Three amino acids are located near the active site (Arg 145, Tyr 248, and Glu 270)

7. 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

8. 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) • 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 compartmentization 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 pathqways.

9. 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

10. Enzymes requiring metal ions as cofactors

11. Many vitamins are coenzyme precursors

12. 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)

13. Each enzyme has aECnumber =EnzymeCommissionnumber • 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 susubclass) • The 4th number is simply a serial number

14. There are six functional classes of enzymes

16. EC Classification Class Subclass Sub-subclass Serial number Enzyme Nomenclature and Classification

17. Example of Enzyme Nomenclature • Common name(s) • Invertase, sucrase • Systematic name • -D-fructofuranoside fructohydrolase (E.C. 3.2.1.26) • Recommended name • -fructofuranosidase

18. Enzyme kinetics Kinetic

19. 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

20. Enzyme Stabilizes Transition State What’s the difference? Many enzymes function by lowering the activation energy of reactions. Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166

21. EA = Activation energy ; a barrier to the reaction Can be overcome by adding energy....... ......or by catalysis

22. 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

23. Active Site Is a Deep Buried Pocket Why energy required to reach transition state is lower in the active site? (1) Stabilizes transition + (2) Expels water CoE (2) (1) (3) Reactive groups (4) - (4) Coenzyme helps (3) Juang RH (2004) BCbasics

24. Active Site Avoids the Influence of Water + - Preventing the influence of water sustains the formation of stable ionic bonds Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.115

25. 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.

26. Factors affecting rate of enzyme reactions • Temperature • pH • Enzyme concentration [E] • Substrate concentration [S] • Inhibition • Regulation (Effectors)

27. 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 C 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

28. 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 intestine (pH 8)

29. 3- Enzyme concentration • The Rate (v) of reaction Increases proportional to the enzyme concentration [E] ([S] is high).

30. 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).

31. Enzyme Velocity Curve 0 1 2 3 4 5 6 7 8 80 60 40 20 0 Product (v) 0 2 4 6 8 Substrate (mmole) [S] S + E P (in a fixed period of time) Constant [E] Juang RH (2004) BCbasics

32. k1 k2 S E k-1 maximal velocity, Vmax 0.5Vmax Km Michaelis-Menten equation S E P

33. MM equation derivation (steady state)

34. 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 • Kcat / Km • Practical idea of the catalytic efficiency, i.e. how often a molecule of substrate that is bound reacts to give product.

35. 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

36. 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 [S] reacting or [P] produced/min. It is better to measure it at linear part of the curve

37. 1 vo vo 1 Vmax - 1 Km 1/S S Lineweaver-Burk plot 1/2 Km Double reciprocal Direct plot Juang RH (2004) BCbasics

38. Allosteric enzymes • Why the sigmoid shape? • Allosteric enzymes are multi-subunit enzymes, each with an active site. • They show a cooperative response to substrates hyperbolic curve michaelis-menten kinetics Sigmoidal curve

39. Irreversible Inhibition = 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

40. 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.

41. The Effects of Enzyme Inhibitors

42. 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.

43. Experiments No Inhibitor Effect of para-hydroxybenzoicacid (PHBA) Effect of phenylthiourea

45. I- Competitive Inhibition EI S E Kic S + E ES E + P + I Kmapp/Vmax Kmapp -1/Kmapp CI Competitive

46. EI ESI S E Kic Kiu S + E ES E + P + + I I 1/Vmaxapp Km/Vmaxapp 0.5Vmax II- Noncompetitive Inhibition NCI Noncompetitive (mixed-type) NCI S E

47. ESI Kiu S + E ES E + P + I 1/Vmaxapp 0.5Vmax Kmapp/Vmaxapp Kmapp -1/Kmapp III- Uncompetitive Inhibition Uncompetitive (catalytic) UCI S E

48. Enzyme inhibitors in medicine • Many current pharmaceuticals are enzyme inhibitors(e.g. HIV protease inhibitors for treatment of AIDS) • An example: Ethanol is used as a competitive inhibitor to treat methanol poisoning. • Methanol formaldehyde (very toxic) • Ethanol competes for the same enzyme. • Administration of ethanol occupies the enzyme thereby delaying methanol metabolism long enough for clearance through the kidneys. Alcohol dehydrogenase

49. Enzymes as diagnostic tools Enzymes can be used as markers for diagnosis and prognosis of disease

50. Some diagnostically important enzymes