ISSN:
1420-908X
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
Abstract Accurate kinetic characterization of stromelysin (MMP-3) inhibitors is critical in the design of potent inhibitors of this enzyme. We have successfully modified a previously described assay [1] which used an internally quenched peptide substrate (Dnp-PYAYWMR) that, upon cleavage by MMP-3, produces the products, Dnp-PYA (quiet) and YWMR (a fluorophore at 360 nm). This improved assay uses purified human MMP-3 in the presence of either 5% methanol or 5% DMSO. Fluorescence intensities associated with total hydrolysis of substrate by enzyme have been successfully mimicked using a combination of the product peptides as a standard. We have determined aK m of 39.2 μM andK cat/K m of 4.6 μM/h for MMP-3 (in 5% MeOH) using this peptide substrate. This assay was used successfully to characterize Ro 31-4724 ((N-[(N-[2-[(N-hydroxycarbamoyl)methyl]-4-methyl-valeryl]-L-leucyl]-L-alanine ethyl ester) as a reversible, tightly binding, inhibitor with aK i of 26 nm.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01972762
