Kinetic assays were prepared by adding 100 μL SB (minus urea) and 100 μL urease (urease from C. ensiformis (Type III powder, 15,000–50,000 units/g (Merck, Germany)) (135 U in PBS, determined above) to a 96-well plate (Corning, New York, USA). To the injector system of the plate reader, SB (plus urea (Merck, Germany)) was added (in excess). Each well was read at 560 nm to achieve cycle time of 6 s, each well was read for a total of ten cycles. The injection system then was replaced with SB containing a different (increasing) concentrations of urea and the plate reader method was run again. To measure parameters in the presence of compound, 100 μL of 2-MA (10 mM, dissolved in SB) was added in place of the original 100 μL of SB.
The Dixon plot was performed with three different concentrations of urea (16.6, 83.3, and 166.5 mM) and with five different concentrations of 2-MA (11.0, 27.4, 54.9, 82.3, and 109.7 mM). The reciprocal of initial rate of reaction was plotted against varying inhibitor concentrations to allow determination of Ki and the mechanism of inhibition.
Curve fitting and statistical analysis was performed using GraphPad Prism (version 7, GraphPad Software Inc., San Diego, CA). Kinetic models (Michaelis–Menten) were fitted using non-linear regression and SEM, 95% confidence intervals and R2 values were assessed.
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