Mass spectrometric analysis of interaction of Streptomyces rimosus lipase with 3, 4-dichloroisocoumarin (CROSBI ID 578532)
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Leščić Ašler, Ivana ; Marchetti-Deschmann, Martina ; Kojić-Prodić, Biserka ; Allmaier, Günter
engleski
Mass spectrometric analysis of interaction of Streptomyces rimosus lipase with 3, 4-dichloroisocoumarin
The regulation of enzyme activity is very important for biochemical research and for applications in various industries. Inhibitors bound in the active site of an enzyme can serve as extremely useful probes to reveal the mechanism of enzyme catalysis. We have identified an extracellular lipase from bacterium Streptomyces rimosus to be the member of the poorly characterized family of SGNH-hydrolases. It has been proposed that these enzymes have the three-dimensional structures and active sites significantly different from other lipases, suggesting a unique catalytic mechanism. Here we describe the mass spectrometric analysis of the inhibition of Streptomyces rimosus lipase (SrLip) with 3, 4-dichloroisocoumarin (DCI). Kinetic analysis proved that the binding is covalent/irreversible via the two-step mechanism. The dissociation constant of the non-covalent E•I complex - Ki*, and first-order rate constant for inactivation - k2, were determined by incubation and progress curve methods. The release of DCI from the active site was followed by measurement of lipase reactivation and by re-appearing of native lipase peak in mass spectra. A half-life of reactivation for lipase inhibited with 10-fold molar excess of DCI was calculated. Also, determination of the active enzyme concentration using inhibition with DCI and subsequent activity and mass spectrometry measurements was performed.
mass spectrometry; Streptomyces rimosus; SGNH-hydrolase; 3; 4-dichloroisocoumarin; inhibition kinetics
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Advances in Metabolic Profiling & Mass Spec Europe
poster
08.11.2011-09.11.2011
Dublin, Irska