Time-course of enzyme-catalyzed competing substrate degradation for michaelian behavior and for enzymes showing activation/inhibition by excess substrate.

Progress curves for competing substrates were analyzed to investigate the effect of an "invisible" substrate (B) on the time-course of enzyme-catalyzed substrate degradation of a "visible" (reporter) substrate (A). Rate equations were integrated for Michaelis-Menten kinetics and in the case of activation or inhibition of degradation of A by excess of substrate B. The shape of progress curves depends on the ratio of specificity constants (kcat/Km)B/A, the competition matrix (R). Mathematical solutions exist for R ≫ 1, R = 1, R ≪ 1. Working at constant reporter substrate A concentration, from the shape of progress curves (sigmoidal or non-sigmoidal), it is possible to define the type of competitor (B), and from the dependence of retardation time (at 90% completion of A, and at inflexion point for sigmoid-like shaped progress curves) on "invisible" substrate B concentration, it is therefore possible to access to catalytic parameters, and/or to titrate enzyme active sites. This competing substrate approach is suitable for investigating new substrates and reversible inhibitors of toxicological and pharmacological interest, investigating enzyme promiscuity, screening of enzymes degrading numerous compounds, and mining new enzymes of medical or biotechnological interest.