بسم الله الرحمن الرحيم

1. بسم الله الرحمن الرحيم

2. Enzymes

3. Objectives • What are enzymes ? • Properties of enzymes • Classification • Factors Affecting Enzyme Action • Enzyme Kinetics • Inhibition of Enzyme Activity • Regulation of Enzyme Activity

4. Criteria معايير of Enzymes They are : • organic catalysts, • proteinin nature, • of high M.W.(molecular weight), • heat labile • present inside the living cells but they can act independent of the cells.

5. Virtually all reactions in the body are mediated by enzymes, • which are protein catalysts that increasethe rate of reactions without being changed in the overall process. • Enzymes direct all metabolic events.

6.  Substrate: Is the organic substance on which the enzyme act. مادة عضوية تتفاعل مع الإنزيم  Product: Is the organic substance produced by the action of the enzyme. المادة الناتجة من عمل الإنزيم  Conzymes: Essential for the activity of enzymes and they are considered تعتبرas second substrates or Co-substrat. ضروري لنشاط الإنزيم

8. Active sites of the enzymes • Enzyme molecules contain a special pocket or cleft called the active site. • The active site contains amino acid side chains that create a three-dimensional surface complementary to the substrate . • The active site binds the substrate,  enzyme–substrate (ES) complex  converted to an enzyme–product (EP) complex that  dissociates to enzyme and product.

9. Nomenclature Each enzyme is assigned two names. • The first is its short, recommended name, convenient for everyday use. • The second is the more complete systematic name, which is used when an enzyme must be identified without ambiguity.

10. Recommended name • Most commonly used enzyme names have the suffix “-ase” attached to the substrate of the reaction (for example, glucosidase, urease, sucrase), or to a description of the action performed (for example, lactate dehydrogenase and adenylyl cyclase). • [Note: Some enzymes retain their original trivial names, which give no hint of the associated enzymic reaction, for example, trypsin and pepsin.]

11. Systematic name • The International Union of Biochemistry and Molecular Biology (IUBMB) developed a system of nomenclature in which enzymes are divided into six major classes (Figure 5.1), each with numerous subgroups. • The suffix -ase is attached to a fairly complete description of the chemical reaction catalyzed, including the names of all the substrates; for example D-glyceraldehyde 3-phosphate:NAD+oxidoreductase.

12. Mode of binding of substrate to Enzymes 2 models for the formation of enzyme-substrate complex: • Rigidmodel of the catalytic site (Lock and key model): • Flexiblemodel of the catalytic Site (induced fit model)

13. Flexiblemodel of the catalytic Site (induced fit model) • The catalytic site is preshaped to fit the substrate. i.e. they are complementary in terms of shape.

14. Rigid model of the catalytic site (Lock and key model): • The substrate induces a conformational change in the catalytic site to fit ininside

16. Enzymes composed wholly of protein are known as simple enzymes • Complex enzymes, (which are composed of  protein plus a relatively small molecule) are known as holoenzymes. • Protein component in the holoenzyme is known as the apoenzyme, while the non-protein component might be coenzymeor cofactor which helps the enzyme to act.

17. Holoenzymes • Some enzymes require molecules other than proteins for enzymic activity. • Holoenzyme = the active enzyme (protein part) + its non-protein component, • apoenzyme = the enzyme without its nonproteinmoiety  and is inactive. • Ifthe nonprotein moiety is a metal ion such as Zn2+ or Fe2+,  called a cofactor. • If it is a small organicmolecule,  it is termed a coenzyme.

18. Holoenzymes • Holoenzyme= apoenzyme+ cofactor or coenzyme Holoenzyme Cofactor or + • Coenzyme apoenzyme

19. Catalytic Efficiency • Most enzyme-catalyzed reactions are highly efficient, proceeding from 103 to 108times faster than uncatalyzed reactions. • Typically, each enzyme molecule is capable of transforming 100 to 1,000 substrate molecules into product each second. • The number of molecules of substrate converted to product per enzyme molecule per second is called the turnover number, or kcat.

20. How do enzymes Work? Enzymes work by weakening bonds which lowers activation energy

21. Virtually 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 that occurs during the formation of product. Enzymes do not initiate the reactions but they inhance them. i.e. enzymes make spontaneous reactions proceed rapidly

23. E.g. Changes in energy during the conversion of a molecule of reactant A to product B as it proceeds through the transition state (high-energy intermediate), T*: A T*  B

24. 6 Major Classes of Enzymes • Oxidoreductase • Transferase • Hydrolase • Lyase • Isomerase • Ligase

26. Oxidoreductase: • catalyze oxidation reduction reactions. • At least one substrate becomes oxidizedand at least one substrate becomes reduced. • Transferase: • Catalyze group transfer reactions- the transfer of a functional group from one molecule to another. • Hydrolase: • In hydrolysis reactions,C-O, C-N, and C-S bondsare cleavedbyaddition of H2Oin the form of OH- and H+ to the atoms forming the bond.

28. Lyase: • LyasescleaveC-C, C-O, C-N, and C-S bonds bymeansother than hydrolysis or oxidation. • Isomerase: • Isomerases just rearrangethe existing atoms of a molecule, that is, create isomers of the starting material. • Ligase: • Ligases synthesizeC-C, C-O, C-N, and C-S bonds in reactions coupledtothe cleavage of high energy phosphate bonds in ATP orsome other nucleotide.

30. Specificity • Enzymes are highly specific • catalyzing only one type of chemical reaction. • interacting with one or a few substrates

31. Enzymes show different levels of specificity which are: Optical specificity Absolute specificity Relative specificity Group specificity

32. Absolute specificity: The enzyme acts on a specific substrate e.g.  Lactase acts on lactose • Optical specificity Most of enzymes in the body have absolute optical specificity e.g. • D - Amino acid oxidase acts only on D-amino acids • L - Amino acid oxidase acts only on  L-amino acids • Group specificity The enzyme acts on a special type of bond e.g.  Pepsin – Trypsin - Aminopeptidase - Carboxypeptidase • Relative specificity: the enzyme acts at different rates onone type of bond in compounds chemically related, e.g. Salivary amylase attacks α 1, 4 glucosidic bonds of starch, glycogen and dextrins

33. Regulation • Enzyme activity can be regulated, that is, enzymes can be activated or inhibited, so • The rate of product formation responds to the needs of the cell.

34. Location within the cell • Many enzymes are localized in specific organelles within the cell . • Such compartmentalizationserves to isolate the reaction substrate or product from other competing reactions. • This provides a favorable environment for the reaction, and organizes the thousands of enzymes present in the cell

36. FACTORS AFFECTING THE RATE OF ENZYME REACTION • Enzyme concentration. • Co-enzymes concentration. • Substrate concentration. • Product concentration. • Temperature. • pH • Physical agents. • Oxidation - reduction. • Activators. • Inhibitors. • Time. • On Studying the effect of one of the previous factors on the rate of enzyme activity, the other factors should be fixed.

37. Substrate Concentration • substrate conc. enzyme activity • until the V max is attained at which the catalytic site of all molecules is completelysaturated with the substrate • after that, any further increase in the substrate concentration will not increase the velocity of the reaction.

38. Michaelis-Mentenequation • It describes how reaction velocity varies with substrate concentration: • The following assumptions are made in deriving the Michaelis-Menten rate equation:

39. Km • Km is numerically equal to the substrate concentration at which the reaction velocity is equal to ½Vmax. Kmdoes not vary with the concentration of enzyme.

40. Effect of Temperature • Optimum temperature: • It is the temperature at which the enzymatic reaction velocity is maximal • It ranges from 37°C to 45°C for mammalian enzymes. • With the increase of temperature,  enzymatic activity increases till 45°C  the enzymatic activity decreases irreversibly due to denaturationof the enzyme. • At 65-70°C complete irreversible loss of enzyme activity occurs.

41. Effect of PH • Optimum pH: Each enzyme has an optimum pH at which appropriate charges are present on both the enzyme and the substrate and the enzyme activity is maximal. • At the extremes of pH the enzyme does not act as it is denatured. • The graph will show a bell shaped curve.

42. Effect of physical agents:  Heating, shaking stirring inhibit enzyme activity by denaturation.  inhibit enzyme activity. Effect of enzymatic activators: ???

43. Enzyme inhibitors • Definition: inhibitors aresubstances that combine with the enzymes competitively ornon competitively leading to decreased catalytic activity of the enzyme. Types

44. Types of Enzymes Inhibitors Competitive (substrate analogue) Reversible non-competitive Irreversible non-competitive

45. Competitive inhibitors • The inhibitor (I) combines with the enzyme (E) at the catalytic site. • The chemical structure of the inhibitor  closely resembles that of the substrate. • It combines “reversibly” with the enzyme forming an Enzyme- Inhibitor complex . • When both the substrate and the inhibitor are present, they compete for the same binding site (catalytic site) on the enzyme surface. • In this type of inhibition the Vmax of the enzyme is not decreased, but the apparent Km is increasedi.e affinity of the enzyme to the substrate. • This type of inhibition is reversible and the inhibition is removed by increasing the substrate concentration.

46. Competitive inhibitors

49. Examples of Competitive inhibitors • Sulfonamides (antibacterials) are similar in structure with the substrate Para amino benzoic acid (PABA) so it competes with it at the catalytic site of enzyme required for synthesis of folic acid (required for formation of nucleic acids required by bacteria for multiplication) leading to its inhibition. • Dicumarol is an oral anticoagulant drug similar in structure with vitamin K so it competes with it at the catalytic site of enzyme leading to inhibition of formation of active prothrombin thus preventing blood from clotting.

50. Reversible non-competitive inhibitors • Combine with the enzyme away from the catalytic site. • Are not similar to substrates in structure. • Can combine with free enzyme or with enzyme substrate complex forming EI or ES-I complex. • They lower the Vmaxof enzyme but donot affect theKm (as the catalytic site is free for combination with the substrate). • e.g. Heavy metal ions (e.g. mercury and lead)