Reversible Enzyme Inhibition - b Identify the term(x or...

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Determination of K I for competitive, uncompetitive, and mixed inhibition 1. Determine v o for several substrate concentrations for each of (at least) three different inhibitor concentrations, and plot 1/vo vs. 1/[S]. 2. Identify the type of inhibition: a. Competitive if three lines intersect at y-intercept, but different x-intercepts for each of the inhibitor concentrations. b. Uncompetitive if three parallel lines; different y- and x-intercepts. c. Mixed : If K I does not equal K I ', x- and y-intercepts are different, lines meet at point above x-axis. c. Noncompetitive . If K I = K I ', three lines intersect at x-intercept, but different y-intercepts. 3. K I is determined from x-intercept, y-intercept, or slope of inverse plot: y = mx + b a.Obtain inverse equation from Michaelis-Menten equation Competitive inhibition v o = Vmax[S] α Km + [S] α = 1 + ([I]/Ki) Uncompetitive Inhibition v o = Vmax[S] Km + α '[S] α ’ = 1 + ([I]/Ki’)
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Mixed inhibition v o = Vmax[S] α Km + α '[S] α = 1 + ([I]/Ki) α ’ = 1 + ([I]/Ki’)
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Unformatted text preview: b. Identify the term (x- or y-intercept, or slope) with K I Example: Mixed inhibition, where K I does not equal K I ' 1) To determine K I , use the slope = (1 + [I]/Ki) Km/Vmax Solve for K I---> K I = [I]/[(Vmax/Km) slope - 1)] 2) To determine K I ', use the y-intercept = (1 + [I]/K I ')/Vmax Solve for KI' ---> KI' = [I]/[(Vmax x y-intercept) –1] 3) Therefore, the Km, Vmax, slope of the line for one of the inhibitor concentrations can be used to determine K I , and the Vmax, y-int. for one of the inhibitor concentrations can be used to determine K I '. c. Obtain expressions for the K I for competitive and uncompetitive inhibition. The following data were obtained on isocitrate lyase from an algae species. Deduce the Km and Vmax for the enzyme and the type of inhibition, and determine the Ki for the inhibitor.-20-10 10 20 30 40 50 100 150 200 250 1/[S], mM-1 1/v, μ M-1 min...
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