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

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 EI complex and first-order rate constant for inactivation were determined by incubation (Ki* = 26.6 ± 2.8 μM, k2 = 12.2 ± 0.6 min–1) or progress curve (Ki* = 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 (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.

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

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