A GENERAL PROGRESS CURVE METHOD FOR THE KINETIC-ANALYSIS OF SUICIDE ENZYME-INHIBITORS

The efficiency of suicide inhibitors is expressed in terms of the kine tic parameters K-I and k(inact) and the partition ratio, which are com monly determined by the well-known dilution assay method. Progress cur ve analysis methods have been widely used in the determination of the kinetic parameters of active-site directed affinity labels and have al so 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 met hod for suicide inhibitors has not been applied to the most common cas e where the progress curve is a result of the turnover of both substra te and inhibitor. In the present study we have attempted to apply this method to a well-characterized suicide inhibitor of dopamine beta-mon ooxygenase (EC 1.14.17.1) where the progress curve is a result of the turnover of both substrate and inhibitor. These results demonstrate th at the kinetic constants determined by this method are highly reproduc ible and are also in excellent agreement with those previously determi ned by the dilution assay method. Efficiency and reproducibility, as w ell as the adaptability of commercially readily available curve-fittin g programs such as Sigma Plot for routine use, are major advantages of this procedure. (C) 1996 Academic Press, Inc.