Chemical mechanism of a cysteine protease, cathepsin C, as revealed by integration of both steady-state and pre-steady-state solvent kinetic isotope effects

Cathepsin C, or dipeptidyl peptidase 1, is a lysosomal cysteine protease of the papain family that catalyzes the sequential removal of dipeptides from the free N-termini of proteins and peptides. Using the dipeptide substrate Ser-Tyr-AMC, cathepsin C was characterized in both steady-state and pre-steady-state kinetic modes. The pH(D) rate profiles for both log k(cat)/K-m and log k(cat) conformed to bell-shaped curves for which an inverse solvent kinetic isotope effect (sKIE) of 0.71 +/- 0.14 for (D)(k(cat)/K-a) and a normal sKIE of 2.76 +/- 0.03 for (D)kat were obtained. Pre-steady-state kinetics exhibited a single-exponential burst of AMC formation in which the maximal acylation rate (k(ac) = 397 +/- 5 s(-1)) was found to be nearly 30-fold greater than the rate-limiting deacylation rate (k(dac) = 13.95 +/- 0.013 s(-1)) and turnover number (k(cat) = 13.92 +/- 0.001 s(-1)). Analysis of pre-steady-state burst kinetics in D2O allowed abstraction of a normal sKIE for the acylation half-reaction that was not observed in steady-state kinetics. Since normal sKlEs were obtained for all measurable acylation steps in the presteady state [(D)k(ac) = 1.31 +/- 0.04, and the transient kinetic isotope effect at time zero (tKIE(0)) = 2.3 +/- 0.2], the kinetic step(s) contributing to the inverse sKIE of D(kcat/Ka) must occur more rapidly than the experimental time frame of the transient kinetics. Results are consistent with a chemical mechanism in which acylation occurs via a two-step process: the thiolate form of Cys-234, which is enriched in D2O and gives rise to the inverse value of (D)(k(cat)/K-a), attacks the substrate to form a tetrahedral intermediate that proceeds to form an acyl-enzyme intermediate during a proton transfer step expressing a normal sKIE. The subsequent deacylation half-reaction is rate-limiting, with proton transfers exhibiting normal sKlEs. Through derivation of 12 equations describing all kinetic parameters and sKlEs for the proposed cathepsin C mechanism, integration of both steady-state and pre-steady-state kinetics with sKlEs allowed the provision of at least one self-consistent set of values for all 13 rate constants in this cysteine protease's chemical mechanism. Simulation of the resulting kinetic profile showed that at steady state similar to 80% of the enzyme exists in an active-site cysteine-acylated form in the mechanistic pathway. The chemical and kinetic details deduced from this work provide a potential roadmap to help steer drug discovery efforts for this and other disease-relevant cysteine proteases.