Enzymes

1. Enzymes • Biological Catalysts • Nomenclature and Classification • Enzyme-Substrate Interaction • Effects of pH and Temperature • Regulation of Enzyme Activity • Cofactors and Coenzymes • Vitamins and Coenzymes

2. Enzymes = Biological catalysts Permit reactions to ‘go’ at body conditions Large proteins pH 7.4, 37oC Process millions of molecules every second Very specific react with only 1 or a few types of molecules (substrates).

3. Effect of enzymes on Eact For all reactions you must get over the activation energy hurdle. Ea Reactants H2O2 Energy H Products H2O + O2

4. Effect of enzymes on Eact Enzyme catalyzed reaction Enzymes change how reactions proceed. Reducing activation energy. Makes faster. Ea Reactants H2O2 Energy H Products H2O + O2

5. Enzyme nomenclature • Name is based on: whatwith or how it reacts + -ase ending Examples To react with lactose. lactase To remove carboxyl from pyruvate. pyruvate decarboxylase

6. Classification of enzymes • Based on type of reaction • Oxireductase • Transferase • Hydrolase • Lyase • Isomerases • Ligase catalyze a redox reaction transfer a functional group catalyze hydrolysis rxns Add or remove to C=C bonds rearrange to form isomers join two molecules

7. The Active Site • Enzymes are typically HUGE proteins, yet only a small part are actually involved in reaction. The active site has two basic components. catalytic site binding site Model of trios-p-isomerase

8. The Active Site Catalytic site Wherereaction occurs Binding Site holds substrate in place Substrate Enzyme

9. SPECIFICITY • Enzymes are very specific. Each enzyme will catalyze only one type of reactions and often will only work with a specific substrate. • Ex. NH2-C-NH2 + H202NH3 + CO2 • Urease has no effect on other compounds. • Such absolute specificity is rather rare among enzymes. urease O

10. Enzyme classes • Absolutely specific • Only reacts with a single substrate. • Group specific • Works with similar molecules with the same functional group. • Linkage specific • Catalyzes a specific combination of bonds. • Stereochemically specific • Only will work with the proper D-or L-form.

11. ISOENZYMES: Different enzymes that perform the same type of function in different organisms or tissues.

12. Enzyme-substrate complex • Step 1: (All of these steps are in equilibrium) • Enzyme and substrate combine to form complex • E + SES • EnzymeSubstrateComplex +

13. Enzyme-product complex • Step 2: • An enzyme-product complex is formed. • ES EP transition state ES EP

14. EP Product • The enzyme and product separate • EPE + P The product is made Enzyme is ready for another substrate.

16. Lock and Key Theory • Enzyme is “lock” and Substrate is the “key”. • Substrate structure • must fit into enzyme’sstructure.

17. Induced Fit Theory • Active site may not fit substrate. • Site must change in order to form the complex.

19. Effect of Temp on Enzymatic Rxns • Exceeding normal pH and temperature ranges always reduces enzyme reaction rates. Optimum Temp usually 37oC. Reaction Rate Temperature

20. Effect of pH on Enzymatic Rxns Most enzymes work best near pH 7.4 not all though. Reaction Rate pH

21. Examples of optimum pH • Optimum • Enzyme Source pH • pepsin gastric mucosa 1.5 • sucrase intestine 6.2 • catalase liver 7.3 • arginase beef liver 9.0 • alkaline bone 9.5 • phosphatase

22. Effect of substrate concentration For non-enzyme catalyzed reactions Rate of reaction (velocity) Rate increases if concentration of the substrate increases Substrate concentration

23. Effect of substrate concentration For Enzyme catalyzed reactions Rates increase but only to a certain point Saturation point Vmax w/ more enzyme Vmax w/ some enzyme AtVmax the enzyme is working as fast as it can. Rate of reaction (velocity) Rate is limited by the concentration of both the substrate and enzyme. Substrate concentration

24. Effect of Enzyme concentration Enzyme Activity Enzyme Concentration

25. Turnover Number • Turnover Number: • The rate at which an enzyme transforms the substrate • Is measured at optimum pH and temperature. • Example: • CarbonicAnhydrase • H2CO3H2O + CO2 • 36,000,000 molecules • minute

26. ENZYME INHIBITION • Inhibitors = interfere with ability of enzyme to react properly with its substrate. • For example: • Medicinal drugs • inhibit by inactivating an enzyme essential to bacterial growth. • Viruses more difficult to inhibit because they use enzyme system of the host cell. • (An inhibitor of a virus also • destroys host cells)

27. ENZYME INHIBITION • Two Types of Inhibitors: • Competitive • Noncompetitive

28. COMPETITIVE INHIBITOR Competes with substrate for the active site. Enzyme mistakes inhibitor for substrate

29. Reversible Competitive inhibition Enzyme - substrate reactions in equilibrium. Inhibitor Substrate EI ES EI I + E + S ES EP  E + P Shifts Inc I Inc S shifts

30. Competitive Inhibitors Sulfa Drugs • Illnesses caused by invading microorganisms like bacterium can be combated using a competitive inhibitor called an antimetabolite. • Folic Acid is a coenzyme in many biosynthetic processes like synthesis of amino acids and nucleotides.

31. Sulfa Drugs • Folic Acid : obtained • from the diet or • from microorganisms in the intestinal tract. Microorganisms makefolic acid fromPABA.

33. Penicillin: War of Enzyme against Enzyme. • Produced by mold, it prevents growth of bacteria by successfully competing for active sites on an enzyme that bacteria need for cell wall production. • 1. Bacteria need the enzyme transpeptidase to make their cell walls rigid and cross-linked. • 2. Penicillin takes control of transpeptidase. • 3. Bacteria cell walls are not cross-linked and the contents of the bacteria cells cannot be held in. • 4. Cytoplasm spills out, and the bacteria die.

34. By changing the R group, science has found a way to prevent this from happening.

35. Non competitive Inhibition • This type of inhibitor is believed to alter the shape of the enzyme and greatly reduce its affinity for the substrate. • 1. It does not compete with the substrate for the active site. • 2. It does not need to resemble the structure of the substrate. • 3. Its’ effect cannot be reversed by increasing the substrate concentration.

36. Non competitive Inhibition • A noncompetitive inhibitor can bind to an enzyme in many ways. • If it binds somewhere on the surface of the enzyme, it causes a change in the tertiary structure. • The substrate is inhibited because it can’t get into the enzyme.

37. Regulation of enzyme activity • Enzymes are often • regulated by the cell. • (Unlike other catalysts) Cells use several methodsto control when & how well enzymes work.

38. PROENZYMES (ZYMOGENS) • Enzymes manufactured in inactive form. Activated when small part of polypeptide chain removed. Hormones, Digestive Enz, Blood Clotting Enz

39. S S S S S S S S S S S S PROENZYMES (ZYMOGENS) Enzymes manufactured in inactive form. In pancreas (inactive) Proinsulin In blood (active) Insulin

40. enteropeptidase PROENZYMES (ZYMOGENS) (inactive) In pancreas (active) In Intestines Trypsinogen Trypsin Chymotrypsinogen Trypsin Chymotrypsin procarboxypeptidase Trypsin Carboxypeptidase Digestive Enzymes Proteases Cleave peptides

41. enteropeptidase PROENZYMES (ZYMOGENS) (inactive) In pancreas (active) In Intestines Trypsinogen Trypsin Chymotrypsinogen Trypsin Chymotrypsin procarboxypeptidase Trypsin Carboxypeptidase • Activation in pancreas rather than intestines  • pancreas proteins get digested • pancreatitis (inflammation of pancreas).

42. H+ PROENZYMES (ZYMOGENS) (inactive) In Gastric mucosa (active) In Stomach Pepsinogen Pepsin Digestive Enzyme HCl Produced as Food enters stomach • As pH  acid • Proenzyme gets cleaved • Pepsin gets activated

43. Allosteric Enzymes • Application of non competitive inhibition • Regulatesaway from active site Inactive Enzyme Active Enzyme Substrate Now fits Positive Regulator Active Site Changed Positive allosterism - activates the enzyme. Negative allosterism - deactivates the enzyme.

44. E1 E2 E3 E1 E1 Inactive Enzyme Feedback Control Negative Regulator End Product Stops E1 B C D A Active Allosteric Enzyme

45. Cofactors • Apoenzyme • protein portion • Inactive Co2+ + Co 2+ Co2+ Cofactor Non protein Group need to ‘activate’ apoenzyme

46. Mineral Cofactors • Metal IonEnzyme involvedFunction • Cu2+ Cytochrome oxidase redox • Fe2+/Fe3+ Catalase redox • Cytochrome oxidase • Zn2+ Alcohol dehydrogenase Used with NAD+ • Mg2+ Glucose-9-phosphatase Hydrolyzes • phosphate esters

47. + apoenzyme coenzyme holoenzyme Coenzymes • Organic molecule that temporarily binds to apoenzyme in order for it to work Non-Protein Total Protein

48. Vitamins are oftenconverted to coenzymes • VitaminCoenzyme madeFunction • B1 thiamine pyrophosphate decarboxylation • B2 flavin mononucleotide carries hydrogen • folic acid tetrahydrofolic acid amino acid • metabolism • biotin biocytin CO2 fixation • pantothenic Coenzyme A acyl group carrier • acid

49. Enzymes in Medical Diagnosis and Treatment • Most enzymes are confined within the cells of the body. • However, small amounts can also be found in body fluids (blood, urine, cerebrospinal fluid) • The level of enzyme activity outside the cells can be easily monitored. • Abnormal activity (high or low) of particular enzymes in various body fluids signals either the onset of certain diseases or their progression.