1 / 40

Enzyme Kinetics - Inhibition

Enzyme Kinetics - Inhibition. Types of Inhibition. Competitive Inhibition Noncompetitive Inhibition Uncompetitive Inhibition Irreversible Inhibition. Competitive Inhibition. In competitive inhibition, the inhibitor competes with the substrate for the same binding site.

allyson
Download Presentation

Enzyme Kinetics - Inhibition

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Enzyme Kinetics - Inhibition

  2. Types of Inhibition • Competitive Inhibition • Noncompetitive Inhibition • Uncompetitive Inhibition • Irreversible Inhibition

  3. Competitive Inhibition In competitive inhibition, the inhibitor competes with the substrate for the same binding site

  4. Competitive Inhibition - Reaction Mechanism In competitive inhibition, the inhibitor binds only to the free enzyme, not to the ES complex

  5. General Michaelis-Menten Equation This form of the Michaelis-Menten equation can be used to understand how each type of inhibitor affects the reaction rate curve

  6. In competitive inhibition, only the apparent Km is affected (Km,app> Km), The Vmax remains unchanged by the presence of the inhibitor.

  7. Competitive inhibitors alter the apparent Km, not the Vmax Vmax,app = Vmax Km,app > Km

  8. The Lineweaver-Burk plot is diagnostic for competitive inhibition

  9. Inhibitor competes with substrate, decreasing its apparent affinity: Km,app > Km Relating the Michaelis-Menten equation, the v vs. [S] plot, and the physical picture of competitive inhibition Km,app > Km Vmax,app = Vmax Formation of EI complex shifts reaction to the left: Km,app > Km Formation of EI complex shifts reaction to the left: Km,app > Km

  10. Example - Competitive Inhibition Sulfanilamide is a competitive inhibitor of p-aminobenzoic acid. Sulfanilamides (also known as sulfa drugs, discovered in the 1930s) were the first effective systemic antibacterial agents. Because we do not make folic acid, sulfanilamides do not affect human cells.

  11. Practical case: Methanol poisoning A wealthy visitor is taken to the emergency room, where he is diagnosed with methanol poisoning. You are contacted by a 3rd year medical student and asked what to do? How would you suggest treating this patient?

  12. Methanol (CH3OH) is metabolized to formaldehyde and formic acid by alcohol dehydrogenase. You advisethe third year student to get the patient very drunk. Since ethanol (CH3CH2OH) competes with methanol for the same binding site on alcohol dehydrogenase, it slows the metabolism of methanol, allowing the toxic metabolites to be disposed of before they build up to dangerous levels. By the way, the patient was very grateful and decided to leave all their worldly possessions to the hospital. Unfortunately, after being released from the hospital, he went to the casinos and lost everything he had.

  13. Noncompetitive Inhibition the inhibitor does not interfere with substrate binding (and vice versa)

  14. Noncompetitive Inhibition - Reaction Mechanism In noncompetitive inhibition, the inhibitor binds enzyme irregardless of whether the substrate is bound

  15. Noncompetitive inhibitors decrease the Vmax,app, but don’t affect the Km Vmax,app < Vmax Km,app = Km

  16. Why does Km,app = Km for noncompetitive inhibition? The inhibitor binds equally well to free enzyme and the ES complex, so it doesn’t alter apparent affinity of the enzyme for the substrate

  17. The Lineweaver-Burk plot is diagnostic for noncompetitive inhibition

  18. Relating the Michaelis-Menten equation, the v vs. [S] plot, and the physical picture of noncompetitive inhibition Inhibitor doesn’t interfere with substrate binding, Km,app = Km Even at high substrate levels, inhibitor still binds, [E]t < [ES] Vmax,app < Vmax Km,app > Km Vmax,app = Vmax Vmax,app < Vmax Km,app = Km Formation of EI complex shifts reaction to the left: Km,app > Km

  19. Noncompetitive inhibitors decrease the apparent Vmax, but do not alter the Km of the reaction

  20. Example of noncompetitive inhibition: fructose 1,6-bisphosphatase inhibition by AMP

  21. Fructose 1,6-bisphosphatase is a key regulatory enzyme in the gluconeogenesis pathway. High amounts of AMP signal that ATP levels are low and gluconeogenesis should be shut down while glycolysis is turned on. High AMP levels inhibit fructose 1,6-bisphosphatase (shutting down gluconeogenesis) and activate phosphofructokinase (turning on glycolysis). Regulation of fructose 1,6-bisphosphatase and phosphofructokinase by AMP prevents a futile cycle in which glucose is simultaneously synthesized and broken down.

  22. Uncompetitive Inhibition In uncompetitive inhibition, the inhibitor binds only to the ES complex

  23. Uncompetitive Inhibition - Reaction Mechanism In uncompetitive inhibition, the inhibitor binds only to the ES complex, it does not bind to the free enzyme

  24. Uncompetitive inhibitors decrease both the Vmax,app and the Km,app Vmax,app < Vmax Km,app < Km Notice that at low substrate concentrations, uncompetitive inhibitors have little effect on the reaction rate because the lower Km,app of the enzyme offsets the decreased Vmax,app

  25. Uncompetitive inhibitors decrease both the Vmax,app and the Km,app of the enzyme Notice that uncompetitive inhibitors don’t bind to the free enzyme, so there is no EI complex in the reaction mechanism

  26. The Lineweaver-Burk plot is diagnostic for uncompetitive inhibition

  27. Relating the Michaelis-Menten equation, the v vs. [S] plot, and the physical picture of uncompetitive inhibition Inhibitor increases the amount of enzyme bound to substrate Km,app < Km Vmax,app < Vmax Km,app< Km Even at high substrate levels, inhibitor binds, [E]t < [ES] Vmax,app < Vmax Formation of EI complex shifts reaction to the left: Km,app > Km

  28. Uncompetitive inhibitors decrease the apparent Km of the enzyme and decrease the Vmax of the reaction

  29. Example of uncompetitive inhibition: alkaline phosphatase inhibition by phenylalanine

  30. At alkaline pH, alkaline phosphatase catalyzes the release of inorganic phosphate from phosphate esters. It is found in a number of tissues, including liver, bile ducts, intestine, bone, kidney, placenta, and leukocytes. Alkaline phosphatase plays a role in the deposition of hydroxyapetite in osteoid cells during bone formation. The function of alkaline phosphatase in other tissues is not known. Serum alkaline phosphatase levels are important diagnostic markers for bone and liver disease.

  31. Irreversible Inhibition In irreversible inhibition, the inhibitor binds to the enzyme irreversibly through formation of a covalent bond with the enzyme , permanently inactivating the enzyme

  32. Irreversible Inhibition - Reaction Mechanism In irreversible inhibition, the inhibitor permanently inactivates the enzyme. The net effect is to remove enzyme from the reaction. Vmax decreases No effect on Km

  33. The Michaelis-Menten plot for an irreversible inhibitor looks like noncompetitive inhibition Vmax,app < Vmax Km,app = Km

  34. Irreversible inhibition is distinguished from noncompetitive inhibition by plotting Vmax vs [E]t Enzyme is inactivated until all of the irreversible inhibitor is used up

  35. Irreversible inhibitors decrease Vmax,app, but leave the apparent Km unchanged. Irreversible inhibitors differ from other types of inhibitors because they covalently modify the enzyme. This results in the permanent inhibition of the enzyme activity.

  36. Examples of Irreversible Inhibitors • diisopropylphosphofluoridate • prototype for the nerve gas sarin • permanently inactivates serine proteases by forming a covalent bond with the active site serine

  37. Penicillin is a suicide inhibitor Glycopeptide transpeptidase catalyzes the formation of cross-links between D-amino acids in the cell walls of bacteria. This enzyme also catalyzes the reverse reaction, the hydrolysis of peptide bonds. During the course of hydrolyzing the strained peptide bond in penicillin, the enzyme activates the inhibitor (penicillin), which then covalently modifies an active site serine in the enzyme. In effect, the enzyme “commits suicide” by hydrolyzing the strained peptide bond in penicillin.

  38. Suicide inhibitors work by “tricking” the enzyme into activating the inhibitor, which then forms a covalent bond with the enzyme, leading to its permanent inactivation.

  39. Summary-Enzyme Inhibition • Competitive Inhibitor • Binds to substrate binding site • Competes with substrate • The affinity of the substrate appears to be decreased when inhibitor is present (Km,app>Km) • Noncompetitive inhibitor • Binds to allosteric site • Does not compete with the substrate for binding to the enzyme • The maximum velocity appears to be decreased in the presence of the inhibitor (Vmax,app <Vmax)

  40. Uncompetitive Inhibitor • Binds to the enzyme only after the substrate has bound • The affinity of the substrate appears to be increased and the maximum velocity appears to be decreased when inhibitor is present (Km,app<Km, Vmax,app <Vmax), • Irreversible Inhibitor • Covalently modifies and permanently inactivates the enzyme

More Related