Insights into the Autoproteolytic Processing and Catalytic
Mechanism of the Chlamydia trachomatis Virulence-Associated
Protease CPAF
Posted on 2019-08-09 - 21:43
CPAF
(chlamydial protease-like activity factor) is a Chlamydia
trachomatis 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 (kcat = 45 ± 0.6 s–1; kcat/Km = 0.37 ± 0.02 μM–1 s–1) or a new fluorescence-based continuous assay (kcat = 23 ± 0.7 s–1; kcat/Km = 0.29 ± 0.03
μM–1 s–1). 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 (kacyl = 110 ± 2 s–1) followed by slower, partially rate-limiting deacylation
(kdeacyl = 57 ± 1 s–1). Both kcat– and kcat/Km–pH profiles
showed single acidic limb ionizations with pKa 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 D2Okcatapp, but a unity effect was found for D2Okcat/Kmapp. The kcat 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.
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Dudiak, Brianne
M.; Maksimchuk, Kenneth R.; Bednar, Maria M.; Podracky, Christopher J.; Burg, Jonathan M.; Nguyen, Tri M.; et al. (2019). Insights into the Autoproteolytic Processing and Catalytic
Mechanism of the Chlamydia trachomatis Virulence-Associated
Protease CPAF. ACS Publications. Collection. https://doi.org/10.1021/acs.biochem.9b00522