Figure 1.
Simulated progress curves and differences between inhibited and uninhibited reactions.
(Left) Simulated progress curves for competitive (blue trace), uninhibited (black trace) reactions, and the difference (Δ[P]) between the two reactions (red trace). Δmax[P] is indicated by a dashed vertical line. (Right) Δ[P] between inhibited and uninhibited enzyme reactions for competitive (yellow), uncompetitive (green), non-competitive (orange), and mixed (blue) inhibition. In the simulation tool, progress curves for all types of inhibition are shown as in the left panel. Reaction parameters and variables can be entered and the results will be directly displayed in the graphs. Entered reaction conditions were: [S] = Km = 0.25Kmp = 10[I] = 400[Eo] = 100 µM; [Po] = 0 µM; kcat = 0.5 s−1; enzyme t(1/2) = 24 hours; Kic = Kiu = Ki-non = 5 µM for competitive, uncompetitive, and non-competitive inhibition; and Kiu = 5Kic = 15 µM for mixed inhibition.
Figure 2.
Inhibition as a function of substrate conversion.
Observed inhibition (%) as a function of the degree of substrate conversion (%) of the uninhibited reference reaction for competitive (yellow), uncompetitive (green), non-competitive (orange), and mixed (blue) inhibition. In the simulation tool, time points can be entered (see Fig. S1) to directly study the effects in the graphs, where observed inhibition is also plotted against reaction time. Entered reaction conditions were: [S] = Km = 0.25Kmp = 10[I] = 400[Eo] = 100 µM; [Po] = 0 µM; kcat = 0.5 s−1; enzyme t(1/2) = 24 hours; Kic = Kiu = Ki-non = 5 µM for competitive, uncompetitive, and non-competitive inhibition; and Kiu = 5Kic = 15 µM for mixed inhibition.
Figure 3.
Observed inhibitor potency (IC'50) as a function of substrate depletion.
The graph shows IC'50 as a function of the degree of substrate conversion of the uninhibited reference reaction for competitive (yellow), uncompetitive (green), non-competitive (orange), and mixed (blue) inhibition. In the tool, the graph is directly coupled to user adjustable reaction variables and parameters. At initial reaction conditions, IC'50 equals IC50. Entered reaction conditions were: [S] = Km = 0.25Kmp = 10[I] = 400[Eo] = 100 µM; [Po] = 0 µM; kcat = 0.5 s−1; enzyme t(1/2) = 24 hours; Kic = Kiu = Ki-non = 5 µM for competitive, uncompetitive, and non-competitive inhibition; and Kiu = 5Kic = 15 µM for mixed inhibition.
Table 1.
Observation at different time points and with different reaction conditions.
Figure 4.
Agreement between simulated and experimental data.
A–B) [P] as a function of time for enzyme catalyzed hydrolysis of alanine-4-nitroanilide by LTA4 hydrolase (A), and of Mca-R-P-P-G-F-S-A-F-K(Dnp)-OH by presequence peptidase (B). Reactions were performed with (green traces) and without (gray traces) the inhibitor bestatin for both enzymes. C–D) Δ[P] as a function of time (orange trace, lower x-axis) and as a function of substrate depletion (gray trace, upper x-axis) for LTA4 hydrolase (C) and for presequence peptidase (D). Simulated curves fit well to the experimental data (thin black lines, all panels). The observed (vertical thick dashed lines) and predicted (vertical thin dashed lines) time point of Δmax[P] are in good agreement. E) Initial reaction rate experiment with presequence peptidase and increasing [S]. The fitted model is shown as a black line, measured data as open circles.
Figure 5.
Screen dump of a subsection of the simulation tool.
The tool contains three blocks (one for competitive, one for uncompetitive, and one for mixed inhibition; non-competitive inhibition is achieved by setting the two Ki values for mixed inhibition to equal values) in which the reaction parameters and variables can be set. The block for mixed inhibition is shown. Adjustable values are shown in red and are found in the second row of each block. The identities of the adjustable values are shown in the top row. The resulting IC50 value and overall equilibrium constant of the reversible reaction are also shown in the top row. A table presents the time point of Δmax[P] and the associated key data that result from adjustment of reaction conditions. To deduce the corresponding data at other time points, the desired values can be entered into cells of the top row of the table. In the simulation tool, changes of reaction conditions are also visualized in various graphs.