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An Internally Quenched Fluorescent Peptide Substrate for Protealysin
Maria A Karaseva1, Ksenia N Chukhontseva1, Irina S Lemeskina1
1Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia.
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.
Insights
Researchers developed a new fluorescent peptide assay for quantifying protealysin-like proteases (PLPs). This assay enables efficient activity studies of PLPs, crucial for understanding bacterial pathogenesis.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- Protealysin is a metalloprotease from Serratia proteamaculans and the prototype of a subgroup within the M4 peptidase family.
- Protealysin-like proteases (PLPs) are prevalent in bacteria, fungi, and archaea, with significant implications for animal and plant pathogenesis.
Purpose of the Study:
- To develop an efficient quantitative assay for measuring the activity of protealysin-like proteases (PLPs).
- To characterize the substrate specificity and kinetic parameters of protealysin and thermolysin using the newly developed assay.
Main Methods:
- A novel internally quenched fluorescent peptide substrate was synthesized based on the autoprocessing site sequence of the protealysin precursor.
- Enzyme kinetics, including Michaelis-Menten constants (KM) and catalytic rates (kcat), were determined for protealysin and thermolysin.
- The substrate's specificity was evaluated against a panel of different proteases.
Main Results:
- The developed substrate specifically detects hydrolysis at the Ser-Val bond by both protealysin and thermolysin.
- Protealysin exhibited a KM of 35 ± 4 μM and kcat of 21 ± 1 s-1, while thermolysin showed a KM of 33 ± 8 μM and kcat of 7 ± 1 s-1.
- While not strictly specific, the substrate demonstrated higher molar activity for protealysin compared to other tested enzymes, including thermolysin.
Conclusions:
- The novel fluorescent peptide substrate provides an effective tool for the quantitative assay of protealysin activity.
- This assay is also valuable for studying the activity of other M4 peptidases, aiding research into bacterial pathogenesis and enzyme mechanisms.
