Inhibition of extracellular lipase from Streptomyces rimosus with 3,4-dichloroisocoumarin

Abstract

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 (K_i* = 26.6 ± 2.8 µM, k₂ = 12.2 ± 0.6 min–1) or progress curve (K_i* = 6.5 ± 1.5 µM, 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 (t_1/2 = 11.3 ± 0.2 h), matrix-assisted laser desorption/ionization (MALDI, t_1/2 = 13.5 ± 0.4 h), and electro-spray ionization (ESI, t_1/2 = 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.

Keywords::

• Enzyme inhibition kinetics
• active-site titration
• mass spectrometry
• SGNH-hydrolase