The amino-acid substituents of dipeptide substrates of cathepsin C can determine the rate-limiting steps of catalysis.

We examined the cathepsin C-catalyzed hydrolysis of dipeptide substrates of the form Yaa-Xaa-AMC, using steady-state and pre-steady-state kinetic methods. The substrates group into three kinetic profiles based upon the broad range observed for kcat/Ka and kcat values, pre-steady-state time courses, and solvent kinetic isotope effects (sKIEs). The dipeptide substrate Gly-Arg-AMC displayed large values for kcat/Ka (1.6 ± 0.09 μM–1 s–1) and kcat (255 ± 6 s–1), an inverse sKIE on kcat/Ka (D(kcat/Ka) = 0.6 ± 0.15), a modest, normal sKIE on kcat (Dkcat = 1.6 ± 0.2), and immeasurable pre-steady-state kinetics, indicating an extremely fast pre-steady-state rate (>400 s–1). (Errors on fitted values are omitted in the text for clarity but may be found in Table 2.) These results conformed to a kinetic model where the acylation (kac) and deacylation (kdac) half-reactions are very fast and similar in value. The second substrate type, Gly-Tyr-AMC and Ser-Tyr-AMC, the latter the subject of a comprehensive kinetic study ...