An Internally Quenched Fluorescent Peptide Substrate for Protealysin

Abstract

Protealysin, a metalloprotease of Serratia proteamaculans, is the prototype of a subgroup of the M4 peptidase family. Protealysin-like proteases (PLPs) are widely spread in bacteria but also occur in fungi and certain archaea. The interest in PLPs is primarily due to their putative involvement in the bacterial pathogenesis in animals and plants. Studying PLPs requires an efficient quantitative assay for their activity; however, no such assay has been reported so far. Here, we used the autoprocessing site sequence of the protealysin precursor to construct an internally quenched fluorescent peptide substrate 2-aminobenzoyl-L-arginyl-L-seryl-L-valyl-L-isoleucyl-L-(ε-2,4-dinitrophenyl)lysine. Protealysin and thermolysin, the prototype of the M4 family, proved to hydrolyze only the Ser-Val bond of the substrate. The substrate exhibited a KM = 35 ± 4 μM and kcat = 21 ± 1 s−1 for protealysin as well as a KM = 33 ± 8 μM and kcat = 7 ± 1 s−1 for thermolysin at 37 °C. Comparison of the effect of different enzymes (thermolysin, trypsin, chymotrypsin, savinase, and pronase E) on the substrate has demonstrated that it is not strictly specific for protealysin; however, this enzyme has higher molar activity even compared to the closely related thermolysin. Thus, the proposed substrate can be advantageous for quantitative studies of protealysin as well as for activity assays of other M4 peptidases.

The role of polymeric chains as a protective environment for improving the stability and efficiency of fluorogenic peptide substrates

“…Thanks to the fluorescent easy understandable and highly specific mechanism, fluorogenic peptide substrates are used to perform fluorometric measurements in various valuable applications, such as studies of binding interactions, and determinations of enzymes’ catalytic activity 15 – 17 . The latter is the most common use, and many examples can be found in the literature, such as determination of catalytic activity of lysostaphin 18 , endopeptidases such as thermolysin, asparaginyl, or protealysin 19 – 21 , and endopeptidase matrix metalloproteases (MMPS) as MMP-2 22 , 23 .…”

Evaluation of the effects of phosphorylation of synthetic peptide substrates on their cleavage by caspase-3 and -7

“…The application of IQF substrates is a common approach used in the examination of the specificity profile of a wide range of proteases, including caspases (34)(35)(36)(37)(38)(39)(40)(41)(42).…”

“Switching On” Enzyme Substrate Specificity Analysis with a Fluorescent Competitive Inhibitor

“…In particular, we can envision that these ideas could be applied to substrate specificity studies of protease and peptidase enzymes. Intramolecular quenching has long been utilized to analyze peptide substrates, and the development of fluorescence-based assay systems is still being investigated. ,, …”

“…Intramolecular quenching has long been utilized to analyze peptide substrates, and the development of fluorescence-based assay systems is still being investigated. 23,33,34 Removed from the principles of intramolecular quenching, existing competitive fluorescence polarization probes could also be fertile ground for "switch on" inhibitors to be used with this transit kinetics approach. Emitting depolarized light when tumbling in solution and polarized light when bound to a larger molecular weight protein, fluorescence polarization probes fit the "switch on" definition required for the transit assay because the signal they emit depends on their binding state.…”