Continuous assays for meprin alpha and beta using prolyl tripeptidyl aminopeptidase (PtP) from Porphyromonas gingivalis
2018
Schulze, Anja | Wermann, Michael | Demuth, Hans-Ulrich | Yoshimoto, Tadashi | Ramsbeck, Daniel | Schlenzig, Dagmar | Schilling, Stephan
Common assays for endoprotease activity of meprin α and β are based on cleavage of internally quenched substrates. Although direct and convenient, for meprins these assays bear disadvantages such as, e.g., significant substrate inhibition or potential fluorescence quenching by compounds applied in inhibitor analysis. Here, we present a novel continuous assay by introducing an auxiliary enzyme, prolyl tripeptidyl aminopeptidase (PtP) and the chromogenic substrate KKGYVADAP-p-nitroanilide. We provide a quick strategy for expression and one-step-purification of the auxiliary enzyme. The enzyme kinetic data for meprin α and β suggest hyperbolic v/S-characteristics, the kinetic parameters of substrate conversion by meprin β were Kₘ = 184 ± 32 μM and kcₐₜ = 20 ± 4 s⁻¹. We also present conditions for the use of the fluorogenic substrate KKGYVADAP-AMC to assess meprin β activity. The assays were applied for determination of inhibitory parameters of the natural inhibitor actinonin and two recently published hydroxamates. Hence, we present two novel methods, which can be applied to assess inhibitory mechanism and potency with the attractive current drug targets meprin α and β. Furthermore, the assay might also provide implications for analysis of other endoproteases as well as their inhibitors.
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