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The molecular mechanisms of competitive and non-competitive enzyme inhibition, including how inhibitors bind to enzymes and the effects on steady-state kinetics. It also covers the analysis of inhibition and the determination of inhibition constants (ki and ki').
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Reading in Campbell: Chapter 6. 19.1 Molecular Mechanisms of Inhibition: A: Competitive Inhibition
Methanol Ethanol
Formaldehyde
Acetaldehyde (^) Acetate
A competitive inhibitor reduces the amount of [E] by the formation of an [EI] complex. The inhibitor cannot affect the [ES] complex after it has formed since the inhibitor can no longer bind. There are two anticipated consequences of this binding mode on the steady-state kinetics:
because some of the enzyme is complexed with inhibitor. B: Non-competitive Inhibition: In this case the inhibitor binds to both [E] and [ES]. The binding site of the inhibitor is not at the active site. However, the inhibitor binding causes a change in the conformation of the protein that affects either substrate binding (Km), the chemical step (Kcat), or both. Both Vmax and Km can be altered by non-competitive inhibitors since the precise geometry of the active site is altered when the inhibitor is bound.
Obtaining KI and KI’ The values of a and a’ can be easily found from the slope and intercept of double reciprocal plots. The five easy steps are:
Obtain a ’ = y-int (+inh) / y-int (-inh): Summary of Kinetic Parameters: Parameter Information content Vmax Information on Kcat – fundamental mechanism Km Information on how tight the substrate binds (but not quite its KD) KI Information on how tight the inhibitor binds to the free enzyme [E] (this is a KD) KI’ Information on how tight the inhibitor binds to the [ES] complex (this is also a KD)
mM v ([I]=0) v ([I]= 1/[S] 1/v ([I]=0) 1/v ([I=10]) 1 16.67 6.25 1.00 0.0600 0. 5 50.00 25.00 0.20 0.0200 0. 20 80.00 57.14 0.05 0.0125 0.