1. ENZYMES ACTIVATION AND DEACTIVATION November 19 th, 2012

2. Enzymes are Not Consumed in Reaction  https://www.youtube.com/watch?v=0XjyAkeQJag& feature=related https://www.youtube.com/watch?v=0XjyAkeQJag& feature=related

3. Factors Effecting Enzymes  Enzymes are not perfect  They respond to environmental conditions  They work depending on various factors which are ?  ________, ___________, _____________  Changing these factors effects ? _____________ Enzyme under Stress

4. pH  An decrease in pH, increases the [H+] ion concentration in solution  An increase in pH, increases the [OH-] ion concentration in solution  These ions interfere with hydrogen bonds and ionic bonds  Changing the conformation of the enzymes specifically the active site  Activity of enzyme is affected

5. Optimal pH  Different enzymes have different optimum pHs  At optimum pH the active site is the shape most complementary to the shape of their Substrate  At optimum pH, the rate of reaction is highest  Large changes in pH can cause enzymes to Denature and permanently loose their function

6. Temperature Effects Enzymes  Generally, enzymes have a narrow range of temperature they work in, Why ?  At the optimal temperature enzymes are most active

7. Increase Temperature  What increases ? energy  Makes the substrate more active in solution. So ?  More chances of substrate colliding with active site.  Makes the enzyme more flexible.  Puts strain on weaker bonds.  Pass a certain point enzymes denature. What does it mean to denature ? active site changes

8. Decrease Temperature  Makes the enzyme less flexible,  Pass a certain point enzymes do not function properly  Not enough energy present

9. Taq Polymerase  Enzyme comes from Thermus Aquaticus, a species that thrive in hot springs and heat vents.  Functions at high temperature  Used in Polymerase Chain Reaction (PCR)  Can make multiple copies of a DNA sample using only a small amount.  PCR can be used for a forensic investigation, genetic diseases, drug discovery and detection of pathogens

10. Temperature Curve  Various thermophillic organisms have their own type of DNA polymerase such as Pfu Polymerase (Pyrococcus furiosus) versus Taq Polymerase

11. Regulation of Temperature  Endotherms can maintain body temperature  Heat is produced and regulated by the body, How ?  Ectotherms do not maintain a body temperature  Less sensitive to changes in body temperature  Endothermic organisms are mammals, birds and some fish  Most enzymes in the human body have an optimal temperature of 37°C

12. Concentration  What is concentration ?  Will the concentration of substrate the rate of reaction ?  Will the concentration of enzyme effect the rate of reaction ?

13. Substrate Concentration  Adding more substrate increases rate of the reaction  More substrate molecule collide with active site  At a certain point adding more substrate has no more effect. Enzyme active site is saturated

14. Enzyme Concentration  If an enzyme is saturated what can you do ?  Increasing enzyme concentration, increases the rate of reaction  Why does the graph level off ?

15. ENZYME REGULATION November 20 th & 21 st, 2012

16. Biochemical Process  http://www.iubmb-nicholson.org/animaps.html http://www.iubmb-nicholson.org/animaps.html

17. Enzyme Regulation  There are enzymes for each specific reaction of the human body  There is a need to control enzyme activity  Regulation is efficiency  Enzymes can be activated and inhibited Road Map

18. Aspirin  Cyclooxygenase 2 (COX2) makes prostaglandins  These chemical are involved in inflammation  Inflammation is felt as pain and swelling in body  Aspirin reacts with the amino acid serine irreversibly, blocking the active site, substrate can not bind  Other pain killers such as ibuprofen (Advil) bind less strongly, are reversible bound

19. Inhibition  Enzyme inhibitors are substances that interfere with catalysis  Inhibitors slow down the rate of reaction  Inhibitors can be reversible or irreversible  Irreversible inhibition – halts enzymatic reaction permanently  Reversible inhibition – slows down the reaction temporarily  Inhibitors can act in a competitive or non competitive form and interfere with the reaction

20. Competitive Inhibition  Competitive inhibition: Enzyme inhibitors prevent the formation of Enzyme-Substrate complexes because they have a similar shape to the substrate molecule.  Prevents enzyme from carrying out reaction it is suited for Enzyme with Active Site Specific for Substrate Substrate Inhibitor Inhibitor Competes with Substrate for the Active Site

21. Competitive Inhibition  Inhibitor has a different shape than the substrate but complements the active site  Inhibitor does not react since it has different structure than the substrate.  Reaction rate is decreased since fewer substrate molecules can bind to the enzyme  Inhibition is typically temporary, the inhibitor eventually leaves the active site  Inhibition depends on the relative concentrations of substrate and inhibitor, both compete for place in enzyme active site

22. Methanol Poisoning  Methanol if ingested is oxidized to formaldehyde and formic acid  Attack on the optic nerve causes blindness.  Methanol found in engine fuel, solvents, window cleaner, and antifreeze Source: http://curriculum.toxicology.wikispaces.net/2.2.5.2.5+Methanol

23. Ethanol Competes with Methanol  Ethanol competitively inhibits the oxidation of methanol by Alcohol Dehydrogenase  Ethanol is oxidized in preference to methanol  Oxidation of methanol is slowed down  Toxic by-products do not have chance to accumulate. Source: http://curriculum.toxicology.wikispaces.net/2.2.5.2.5+Methanol

24. Pennicillin  Pennicillin, an antibiotic, works against disease causing bacteria  Stops cell wall cross-linking permanently  Inactivates transpeptidase, used to build cross-linked peptidoglycan layer in the membrane  The cross-linking peptide chains have repeats of D-Alanine  Pennicillin also has a repeat of D-Alanine-D-Alanine E.Coli cells can not grow and die

25. Succinate Dehydrogenase Inhibitor  Succinate Dehydrogenase catalyzes the conversion of succinate to fumerate, an important biochemical reaction in cellular respiration.  Malonate inhibits this reaction competitively  Used to find active site chemistry  Used to study inborn errors of metabolism

26. Non-Competitive Inhibition  Non-competitive inhibition: enzyme inhibitors prevent the formation of Enzyme-Product Complexes.  Inhibitors prevent the substrate to react and form into product  Non-competitive inhibitors bind to a site other than the Active Site  Binding causes conformational changes that change the tertiary structure of the enzyme  Thus, enzyme can not catalyze reaction

27. Non-Competitive Inhibition Substrate Non-competitive Inhibitor Enzyme Active Site Complementary to Substrate

28. Non-Competitive Reaction  Since they do not compete with substrate molecules, non-competitive inhibitors are not affected by substrate concentration.  Many non-competitive inhibitors are irreversible and permanent, and effectively denature the enzymes which they inhibit.  However, there are a lot of non-permanent and reversible non-competitive inhibitors that are vital in controlling metabolic functions in organisms.

29. Cyanide Poisoning  Another enzyme found in cellular respiration is cytochrome oxidase, one of the most important enzymes in the electron transport chain of reactions that occurs in the mitochondria inner membrane  Here oxygen is reduced and 34 ATP molecules are made.

30. Cyanide Poisoning  Cyanide acts as a non-competitive inhibitor for cytochrome oxidase complex  Cyanide does not compete for the active sites of the enzyme because it has no similarity to the substrate cytochrome  Cyanide attaches to another site on the enzyme and disrupts the enzyme's shape.  This brings the electron transport chain to a halt  No energy can be derived out of respiration  Hydrogen cyanide inhibits metal-containing enzymes in the body, such as cytochrome c-oxidase, which contains iron

31. Irons in Enzymes  Chemical catalyst are usually metals  Many enzymes get their ability to catalyze reactions due to metals found in the active site  One common metal used is iron Fe 2+ that is found in a protoporphyrin ring

32. Ferrochelatase  Ferrochetalase inserts iron into protoporphyrin rings  Lead forms covalent bonds with the sulphydryl side chains of the amino acid cysteine in the enzyme and prevents catalytic activity  The binding of the heavy metal shows non- competitive inhibition because the substrate still has access.

33. Chymotrypsin  Chymotrypsin is an enzyme which hydrolyzes peptides bonds  In its active site there are three amino acids Histidine57, Serine195 and Asparagine102 known.

34. Hydrogen Ion Inhibits Chymotrypsin  These amino acids allow for the substrate to be cleaved.  By lowering pH, amino acids in the active site no longer accept hydrogen proton since Asp102 becomes protonated (hydrogens added)  Hydrogen ion acts as a non-competitive inhibitor by preventing catalysis but do not prevent the substrate from binding to the active site.

35. Biochemical Pathway  A biochemical pathway is a series of step reactions leading to a product  Enzymes lie in biochemical pathways  There are specific enzymes for each reaction step  Metabolism is a sum of biochemical pathways and is made of anabolic and catabolic processes

37. Need to Regulate  There are so many pathways that are incorporated in the metabolic system of the human body  An efficient process is needed to regulate the use of resources and ensure that only what is required is being produced or broken down  Enzymes can be regulated by the ability to be activated and deactivated when needed

38. Allosteric Enzyme Regulation  An allosteric site, a site away from the active site, can bind molecules to change conformation of the enzyme.  At the allosteric site for an inhibitor, binding of an inhibitor causes a conformational change such that the active sites of an enzyme are non complementary to the substrate.  An activator can bind to its allosteric site to open or improve the fit between substrate and enzyme.

40. Feedback Inhibition  In a biochemical pathway, by controlling an earlier step, the next series of reaction steps can be controlled  Usually the end product in a chain of reactions is an inhibitor of an earlier enzyme in the chain to stop the creation of more product  Process is self-regulating and cell resources are not wasted by making more product than needed

41. Feedback Inhibition Using an Allosteric Site

42. Regulation of Glycolysis  Glycolysis is biochemical process where glucose is broken down to pyruvate  Pyruvate is used in mitochondria in the process of aerobic respiration to derive ATP  Pyruvate kinase is the enzyme that converts phosphenolpyruvate to pyruvate in glycolysis  This enzyme is the third regulated enzyme of glycolysis  ATP and alanine act as allosteric inhibitors of pyruvate kinase

44. Feedback Inhibition of Pyruvate