Enzymes

1. Enzymes Dr.Wesal.A.ElHanbli

2. All reactions in the body are mediated by enzymes, which are: • (protein catalysts that increase the rate of reactions without being changed in the overall process.)

3. NOMENCLATURE • Most commonly used enzyme names have the suffix “-ase” attached to the substrate of the reaction eg, glucosidase and urease, • or to a description of the action performed eg, lactate dehydrogenase . But… • trypsin and pepsin.

4. the International Union of Biochemists (IUB) developed a complex but unambiguous system of enzyme nomenclature. • Each enzyme has a code (EC) of 4 digits: 1st digit is for the class, 2nd one for the subclass, 3rd digit for the sub-subclass and the 4th one denotes the specific enzyme, e.g., 2.7.1.1. • EC is used for research purposes.

5. enzymes are divided into six major classes

6. Class 1:Oxidoreductases • Catalyze oxidation-reduction reactions • e.g., alcohol: NAD+ oxidoreductase (alcohol dehydrogenase)

7. Subclasses: -Dehydrogenases: remove two electrons and two H atoms from the substrate -Oxidases: transfer two electrons from the donor to oxygen resulting usually in H2O2 e.g. glucose oxidase. -Oxygenases: add oxygen into a substrate

8. -Peroxidases: utilizes H2O2 rather than oxygen as the oxidant e.g., NADH peroxidase catalyzes the reaction NADH + H+ + H2O2 → NAD+ + 2H2O -Catalase: H2O2 acts as donor and acceptor. Catalase function in the cell to detoxify H2O2 H2O2 + H2O2 → O2 + 2H2O

9. Class 2:Transferases • These enzymes transfer functional groups between substrates • e.g., for subclasses: phosphotransferases(kinases) transfer phosphate groups like glucokinase or hexokinase • glucose + ATP → glucose 6-phosphate • Aminotransferases transfer amino groups

11. Class 3:Hydrolases • Enzymes which catalyze hydrolytic cleavage e.g., peptidases

12. Class 4: Lyases • Add or remove the elements of water, ammonia, or CO2 e.g., dehydratases remove H2O in a dehydration reaction

13. Class 5: Isomerases • Catalyze isomerizations of different types • Sublasses: epimerases, racemases, isomerases, mutases

14. Class 6: Ligases • Catalyze joining of two molecules using energy (ATP). They are called synthetases since they are involved in synthetic reactions

15. PROPERTIES OF ENZYMES • 1. Active sites • Enzyme molecules contain a special pocket or cleft called the active site (substrate binding). • 2.Catalytic efficiency • Enzyme-catalyzed reactions are highly efficient, proceeding from 10³–108 times faster than uncatalyzed reactions.

16. 3. Specificity • Enzymes are highly specific, interacting with one or a few substrates and catalyzing only one type of chemical reaction. • 4. Holoenzymes • The term holoenzyme refers to the active enzyme with its nonprotein component,

17. whereas the enzyme without its nonprotein moiety is termed an apoenzyme and is inactive. • If the nonproteinmoiety is a metal ion such as Zn2+ or Fe2+, it is called a cofactor. • If it is a small organic molecule, it is termed a coenzyme.

18. If the coenzyme is permanently associated with the enzyme and returned to its original form, it is called a prosthetic group eg:. B6 and biotin. • 5. Regulation • Enzyme activity can be regulated, that is, increased or decreased. • 6. Location within the cell • Many enzymes are localized in specific organelles within the cell.

19. FACTORS AFFECTING REACTION VELOCITY • A. Substrate concentration • The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached (enzyme saturation).

20. B. Temperature • The reaction velocity increases with temperature until a peak velocity is reached. • Further elevation of the temperature results in a decrease in reaction velocity due to denaturation of the enzyme. • The optimum temperature for most human enzymes is between 35 and 40°C.

22. C. pH • Effect of pH on the ionization of the active site • The pH optimum varies for different enzymes • Extremes of pH can also lead to denaturation of the enzyme

24. Allosteric enzymes • Allosteric enzymes are usually composed of multiple subunits, • are regulated by molecules called effectors ( modifiers) that bind non-covalently at a site other than the active site, called (allosteric) site .

25. Most enzymes show Michaelis-Menten kinetics, in which the plot of initial reaction velocity (vo) against substrate concentration ([S]), is hyperbolic. • In contrast, allosteric enzymes do not follow Michaelis-Menton kinetics and show a sigmoidal curve

27. ENZYMES EMPLOY MULTIPLE MECHANISMS TO FACILITATE CATALYSIS • Catalysis by Proximity • Acid-Base Catalysis • Catalysis by Strain • Covalent Catalysis

28. SUBSTRATES INDUCE CONFORMATIONAL CHANGES IN ENZYMES • There are two models to explain the specificity of the enzyme to its substrate: A. Lock and key model: • The active site is pre-shaped • This model gives a rigid picture of the enzyme and cannot account for effects of allostericligands.

29. B. Induced-fit model: • A more flexible model in which the binding and active sites are not fully preformed. • Interaction of the substrate and the enzyme induces a conformational change in the enzyme resulting in the formation of stronger binding site.

30. Energy changes occurring during the reaction: • all chemical reactions have an energy barrier separating the reactants and the products. • This barrier, called the free energy of activation, is the energy difference between that of the reactants and a high-energy intermediate (the transition state) that occurs during the formation of product.

32. Rate of reaction: • For molecules to react, they must contain sufficient energy to overcome the energy barrier of the transition state. • In the absence of an enzyme, only a small proportion of a population of molecules may possess enough energy to achieve the transition state between reactant and product.

33. In general, the lower the free energy of activation, the more molecules have sufficient energy to pass through the transition state, and, thus, the faster the rate of the reaction.

34. MICHAELIS-MENTEN EQUATION

35. Important conclusions about Michaelis-Menten kinetics • 1. Characteristics of Km • Km is numerically equal to the substrate concentration at which the reaction velocity is equal to 1⁄2Vmax.

36. It reflects the affinity of the enzyme for that substrate. • small (low) Km reflects a high affinity of the enzyme for substrate because a low concentration of substrate is needed to half-saturate the enzyme—that is, to reach a velocity that is 1⁄2Vmax. • large (high) Km reflects a low affinity of enzyme for substrate.

38. 2. Relationship of velocity to enzyme concentration: • The rate of the reaction is directly proportional to the enzyme concentration at all substrate concentrations.

39. Lineweaver-Burk plot (called a double-reciprocal plot) • It’s the linear form of Michaelis-Menten equation • can be used to; • calculate Km and Vmax, • to determine the mechanism of action of enzyme inhibitors.

40. Lineweaver-Burk plot

41. INHIBITION OF ENZYME ACTIVITY • an inhibitor is substance that can diminish the velocity of an enzyme-catalyzed reaction. Which can be: • 1. Reversible inhibitors typically bind to enzymes through non-covalent bonds. • 2. Irreversible inhibitors bind to enzymes through covalent bonds.

42. Reversible inhibitors: • Types of reversible inhibition are: • competitive and • noncompetitive.

43. Competitive inhibition • competes with the substrate for the active site. • This effect isreversed by increasing [S].eg: • Statin drugs

44. It shows a characteristic Lineweaver-Burk plot in which the plots of the inhibited and uninhibited reactions intersect on the y-axis at 1/Vmax (Vmax is unchanged). • The inhibited and uninhibited reactions show different x-axis intercepts, indicating that the apparent Km is increased in the presence of the competitive inhibitor.

46. Noncompetitive inhibition • occurs when the inhibitor and substrate bind at different sites on the enzyme. • is recognized by its characteristic effect on Vmax. decrease the apparent Vmax • do not interfere with the binding of substrate to enzyme. Thus, no change in Km. • Noncompetitive inhibition can be reversed by dialysis.

48. Irreversible inhibitors: • Some inhibitors act by forming covalent bonds with specific groups of enzymes.eg • lead (Ferrochelatase in heme synthesis) • Insecticides • Hydrogen cyanide

49. REGULATION OF ENZYME ACTIVITY • an increase in substrate concentration prompts an increase in reaction rate. • some enzymes with specialized regulatory functions respond to: • 1.Allosteric control or • 2. Covalent modification, or • 3. Induction and repression

50. Allostericenzymes • are regulated by molecules called effectors ( modifiers) that bind non-covalently at a site other than the active site, called (allosteric) site . • Effectors that inhibit enzyme activity are termed negative effectors, whereas those that increase it are called positive effectors.