Insights into the Autoproteolytic Processing and Catalytic Mechanism of the Virulence-Associated Protease CPAF.

CPAF (chlamydial protease-like activity factor) is a protease that is translocated into the host cytosol during infection. CPAF activity results in dampened host inflammation signaling, cytoskeletal remodeling, and suppressed neutrophil activation. Although CPAF is an emerging antivirulence target, its catalytic mechanism has been unexplored to date. Steady state kinetic parameters were obtained for recombinant CPAF with vimentin-derived peptide substrates using a high-performance liquid chromatography-based discontinuous assay ( = 45 ± 0.6 s; / = 0.37 ± 0.02 μM s) or a new fluorescence-based continuous assay ( = 23 ± 0.7 s; / = 0.29 ± 0.03 μM s). Residues H105, S499, E558, and newly identified D103 were found to be indispensable for autoproteolytic processing by mutagenesis, while participation of C500 was ruled out despite its proximity to the S499 nucleophile. Pre-steady state kinetics indicated a burst kinetic profile, with fast acylation ( = 110 ± 2 s) followed by slower, partially rate-limiting deacylation ( = 57 ± 1 s). Both - and /-pH profiles showed single acidic limb ionizations with p values of 6.2 ± 0.1 and 6.5 ± 0.1, respectively. A forward solvent deuterium kinetic isotope effect of 2.6 ± 0.1 was observed for , but a unity effect was found for /. The proton inventory was linear, indicating transfer of a single proton in the rate-determining transition state, most likely from H105. Collectively, these data provide support for the classification of CPAF as a serine protease and provide a mechanistic foundation for the future design of inhibitors.