title: 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 (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.
authors.affiliation: Rudjer Bošković Institute, Department for Physical Chemistry , Zagreb , Croatia.
authors.forename: Ivana Leščić
authors.initials: IL
authors.lastname: Ašler
authors.forename: Filip
authors.initials: F
authors.lastname: Kovačić
authors.forename: Martina
authors.initials: M
authors.lastname: Marchetti-Deschmann
authors.forename: Günter
authors.initials: G
authors.lastname: Allmaier
authors.forename: Zoran
authors.initials: Z
authors.lastname: Stefanić
authors.forename: Biserka
authors.initials: B
authors.lastname: Kojić-Prodić
mesh_terms: D003374:Coumarins / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D005110:Extracellular Space / Q000187:drug effects / Q000201:enzymology*; D049934:Isocoumarins; D008049:Lipase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015394:Molecular Structure; D013302:Streptomyces / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D013329:Structure-Activity Relationship
chemical_list: D003374:Coumarins; D004791:Enzyme Inhibitors; D049934:Isocoumarins; C046235:3,4-dichloroisocoumarin; D008049:Lipase