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Enzyme Kinetics - Inhibition
Types of InhibitionCompetitive InhibitionNoncompetitive InhibitionUncompetitive InhibitionIrreversible Inhibition
Competitive Inhibition In competitive inhibition, the inhibitor competes with the substrate for the same binding site
Competitive Inhibition - Reaction Mechanism In competitive inhibition, the inhibitor binds only to the free enzyme, not to the ES complex
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
In competitive inhibition, only the apparent Km is affected (Km,app> Km), The Vmax remains unchanged by the presence of the inhibitor.
Competitive inhibitors alter the apparent Km, not the VmaxVmax,app = VmaxKm,app > Km
The Lineweaver-Burk plot is diagnostic for competitive inhibition
Relating the Michaelis-Menten equation, the v vs. [S] plot, and the physical picture of competitive inhibitionFormation of EIcomplex shifts reactionto the left: Km,app > KmKm,app > KmVmax,app = VmaxFormation of EIcomplex shifts reactionto the left: Km,app > Km
Example - Competitive InhibitionSulfanilamide 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.
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?
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.
Noncompetitive Inhibition the inhibitor does not interfere with substrate binding (and vice versa)
Noncompetitive Inhibition - Reaction Mechanism In noncompetitive inhibition, the inhibitor binds enzyme irregardless of whether the substrate is bound
Noncompetitive inhibitors decrease the Vmax,app, but dont affect the KmVmax,app < VmaxKm,app = Km
Why does Km,app = Km for noncompetitive inhibition?The inhibitor binds equally well to free enzyme and the ES complex, so it doesnt alter apparent affinity of the enzyme for the substrate
The Lineweaver-Burk plot is diagnostic for noncompetitive inhibition
Relating the Michaelis-Menten equation, the v vs. [S] plot, and the physical picture of noncompetitive inhibitionFormation of EIcomplex shifts reactionto the left: Km,app > KmKm,app > KmVmax,app = VmaxInhibitor doesnt interferewith substrate binding,Km,app = KmEven at highsubstrate levels,inhibitor still binds,[E]t < [ES]Vmax,app < Vmax Vmax,app < Vmax
Km,app = Km
Noncompetitive inhibitors decrease the apparent Vmax, but do not alter the Km of the reaction
Example of noncompetitive inhibition: fructose 1,6-bisphosphatase inhibition by AMP
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.
Uncompetitive Inhibition In uncompetitive inhibition, the inhibitor binds only to the ES complex
Uncompetitive Inhibition - Reaction Mechanism In uncompetitive inhibition, the inhibitor binds only to the ES complex, it does not bind to the free enzyme
Uncompetitive inhibitors decrease both the Vmax,app and the Km,appVmax,app < VmaxKm,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
Uncompetitive inhibitors decrease both the Vmax,app and the Km,app of the enzyme Notice that uncompetitive inhibitors dont bind to the free enzyme, so there is no EI complex in the reaction mechanism
The Lineweaver-Burk plot is diagnostic for uncompetitive inhibition
Relating the Michaelis-Menten equation, the v vs. [S] plot, and the physical picture of uncompetitive inhibitionFormation of EIcomplex shifts reactionto the left: Km,app > KmEven at highsubstrate levels,inhibitor binds,[E]t < [ES]Vmax,app < Vmax Inhibitor increasesthe amount of enzyme boundto substrateKm,app < KmVmax,app < Vmax
Uncompetitive inhibitors decrease the apparent Km of the enzyme and decrease the Vmax of the reaction
Example of uncompetitive inhibition: alkaline phosphatase inhibition by phenylalanine
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.
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
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
The Michaelis-Menten plot for an irreversible inhibitor looks like noncompetitive inhibitionVmax,app < VmaxKm,app = Km
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
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.
Examples of Irreversible Inhibitorsdiisopropylphosphofluoridateprototype for the nerve gas sarinpermanently inactivates serine proteases by forming a covalent bond with the active site serine
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.
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.
- Summary-Enzyme InhibitionCompetitive InhibitorBinds to substrate binding site Competes with substrate The affinity of the substrate appears to be decreased when inhibitor is present (Km,app >Km)Noncompetitive inhibitorBinds to allosteric siteDoes not compete with the substrate for binding to the enzymeThe maximum velocity appears to be decreased in the presence of the inhibitor (Vmax,app
- Uncompetitive InhibitorBinds to the enzyme only after the substrate has boundThe affinity of the substrate appears to be increased and the maximum velocity appears to be decreased when inhibitor is present (Km,app