Scheme 4.
Partial competitive inhibition; α > 1
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α Is a ratio of the inhibitor’s affinity for the free enzyme vs. its affinity for the enzyme–substrate complex and is described by Eq. (1),
equation(1)Slope=∞=αKmVmaxwhere = ∞ is the lowest concentration of inhibitor where the slope of the double reciprocal plots no longer changes. To determine whether the slope at [1e] = 100 μM was the limiting slope, kinetic assays were performed in the presence of 2, 3, and 4 mM concentrations of 1e (data not shown). Since, the slopes of the reciprocal plots at these z vad fmk three concentrations were identical to desmosome determined at [1e] = 100 μM, the slope at 100 μM 1e was taken as the limiting slope value (Slope=∞).
Using Eq. (1) and a limiting slope value of 9 ± 1 min, a Vmax of 51 ± 2 μM min?1 and Km of 146 ± 8 μM, α is calculated to be 3.1 ± 0.4, indicating that the affinity of 1e for the free enzyme is three times greater than for the enzyme–substrate complex. The Ki for 1e was calculated from Eq. (2). The slope of each line at intermediate concentrations of 1e in Fig. 1 is described by Eq. (2). Therefore, using a value of 5.9 ± 0.4 min for the slope of the line when [1e] = 20 μM and the previously mentioned values for α, Vmax, and Km, Ki equals 6 ± 2 μM.
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