Real-time measurements of dark substrate catalysis

We have developed a novel procedure to monitor the real-time cleavage of na tural unmodified peptides (dark substrates). Tn the competition-based assay , the initial cleavage rate of a fluorogenic peptide substrate is measured in the presence of a second substrate that is not required to exhibit any o ptical property change upon cleavage. Using a unique experimental design an d steady-state enzyme kinetics for a two-substrate system, we were able to determine both K-m and k(cat) values for cleavage of the dark substrate. Th e method was applied to HIV-1 protease and to the V82F/I84V drug resistant mutant enzyme. Using two different substrates, we showed that the kinetic p arameters derived from the competition assay are in good agreement with tho se determined independently using standard direct assay. This method can be applied to other enzyme systems as long as they have one substrate for whi ch catalysis can be conveniently monitored in real time.