Continuous assays for meprin alpha and beta using prolyl tripeptidyl aminopeptidase (PtP) from Porphyromonas gingivalis.
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.
['Schulze|Anja|A|', 'Wermann|Michael|M|', 'Demuth|Hans-Ulrich|HU|', 'Yoshimoto|Tadashi|T|', 'Ramsbeck|Daniel|D|', 'Schlenzig|Dagmar|D|', 'Schilling|Stephan|S|']
D001426:Bacterial Proteins D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases D004305:Dose-Response Relationship, Drug D006877:Hydroxamic Acids D007700:Kinetics D008666:Metalloendopeptidases D015394:Molecular Structure D016966:Porphyromonas gingivalis D011480:Protease Inhibitors D012697:Serine Endopeptidases D013329:Structure-Activity Relationship
Continuous assay Prolyl tripeptidyl aminopeptidase (PtP) Auxiliary enzyme Astacins Meprin α/β