Asler_2013_J.Enzyme.Inhib.Med.Chem_28_1094

Kinetic characterization of lipase inhibition was performed by activity measurement and mass spectrometry (MS), for the first time with serine-protease inhibitor 3,4-dichloroisocoumarin (DCI). Inhibition of Streptomyces rimosus extracellular lipase (SrLip), a member of the SGNH superfamily, by means of DCI follows the mechanism of two-step irreversible inhibition. The dissociation constant of the noncovalent E*I complex and first-order rate constant for inactivation were determined by incubation (Ki* = 26.6 +/- 2.8 microM, k2 = 12.2 +/- 0.6 min-1) or progress curve (Ki* = 6.5 +/- 1.5 microM, k2 = 0.11 +/- 0.01 min-1) method. Half-times of reactivation for lipase inhibited with 10-fold molar excess of DCI were determined by activity measurement (t1/2 = 11.3 +/- 0.2 h), matrix-assisted laser desorption/ionization (MALDI, t1/2 = 13.5 +/- 0.4 h), and electro-spray ionization (ESI, t1/2 = 12.2 +/- 0.5 h) MS. The active SrLip concentration was determined by incubating the enzyme with near equimolar concentrations of DCI, followed by activity and MS measurement.