Chemical Mechanism of a Cysteine Protease, Cathepsin C, As Revealed by Integration of both Steady-State and Pre-Steady-State Solvent Kinetic Isotope Effects

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• 作者：Jessica L. Schneck ; James P. Villa ; Patrick McDevitt ; Michael S. McQueney ; Sara H. Thrall ; Thomas D. Meek
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：August 19, 2008
• 年：2008
• 卷：47
• 期：33
• 页码：8697-8710
• 全文大小：398K
• 年卷期：v.47,no.33(August 19, 2008)
• ISSN：1520-4995

文摘

Cathepsin C, or dipeptidyl peptidase I, 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 ^Dk_cat 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 sKIEs were obtained for all measurable acylation steps in the presteady state [^Dk_ac = 1.31 ± 0.04, and the transient kinetic isotope effect at time zero (tKIE⁰) = 2.3 ± 0.2], the kinetic step(s) contributing to the inverse sKIE of ^D(k_cat/K_a) 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 sKIEs. Through derivation of 12 equations describing all kinetic parameters and sKIEs for the proposed cathepsin C mechanism, integration of both steady-state and pre-steady-state kinetics with sKIEs 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 ∼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.