Abstract:
The efficiency of suicide inhibitors is expressed in terms of the kinetic parameters KI and kinact and the partition ratio, which are commonly determined by the well-known dilution assay method. Progress curve analysis methods have been widely used in the determination of the kinetic parameters of active-site-directed affinity labels and have also been applied to a few suicide inhibitors where the turnover rate of the regular substrate could be measured independently of the turnover rate for the inhibitor, when both the substrate and the inhibitor are present in the assay medium. However, the progress curve analysis method for suicide inhibitors has not been applied to the most common case where the progress curve is a result of the turnover of both substrate and inhibitor. In the present study we have attempted to apply this method to a well-characterized suicide inhibitor of dopamine beta-monooxygenase (EC 1.14.17.1) where the progress curve is a result of the turnover of both substrate and inhibitor. These results demonstrate that the kinetic constants determined by this method are highly reproducible and are also in excellent agreement with those previously determined by the dilution assay method. Efficiency and reproducibility, as well as the adaptability of commercially readily available curve-fitting programs such as Sigma Plot for routine use, are major advantages of this procedure.