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Determination of the catalytically active serine of extracellular lipase from Streptomyces rimosus by inhibition studies and mass spectrometry (CROSBI ID 497339)

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Kovačić, Filip ; Leščić, Ivana ; Zehl, Martin ; Abramić, Marija ; Allmaier, Günter ; Kojić-Prodić, Biserka Determination of the catalytically active serine of extracellular lipase from Streptomyces rimosus by inhibition studies and mass spectrometry. Zagreb, 2004

Podaci o odgovornosti

Kovačić, Filip ; Leščić, Ivana ; Zehl, Martin ; Abramić, Marija ; Allmaier, Günter ; Kojić-Prodić, Biserka

engleski

Determination of the catalytically active serine of extracellular lipase from Streptomyces rimosus by inhibition studies and mass spectrometry

Lipases (EC 3.1.1.3) are enzymes that have important role in biological systems but also have wide industrial applications. They catalyze both hydrolysis and synthesis of esters depending on reaction conditions. All lipases with solved 3D structure share common structural motif (alpha/beta-hydrolase fold) and catalytic triad (Ser, Asp, His) similar to the one of serine proteases. In this study inhibitory activity of phenylmethylsulfonyl-fluorid (PMSF) and 3, 4-dichloroisocoumarin (DCI), representing typical inhibitor agents for serine proteases, and tetrahydrolipstatin (THL), pancreatic lipase inhibitor, was investigated on extracellular lipase from Streptomyces rimosus (SrL), and determination of the catalytically active serine with mass spectrometry (MS) is shown. Incubation of SrL with different concentrations of PMSF did not result in decrease of lipolytic activity. Addition of potential activators of lipases like organic solvents (1, 4-dioxan, 2-propanol) and surfactant (n-octyl-ß-glucopyranoside) before adding PMSF did not significantly influence inhibition behavior of PMSF towards SrL. DCI and THL both inactivated Streptomyces rimosus lipase. DCI (30-fold molar excess) almost completely inhibited SrL already after 15 min of incubation. However, nearly total inhibition of SrL with THL (5560-fold molar excess) was obtained only after 24 h incubation in the presence of 50% 2-propanol. The inactivation kinetics of SrL by THL and DCI was monitored and kinetic parameters were determined. DCI-inhibited Streptomyces rimosus lipase was analyzed with mass spectrometry. MALDI-MS of intact SrL-DCI complex showed one inhibitor molecule bound per lipase molecule. The complex was subjected to tryptic digestion and peptide mass fingerprints were recorded. The peptide containing covalently bound DCI was identified and further analyzed with tandem MS. Catalytic serine (with bound inhibitor) was proven to be Ser10.

Streptomyces rimosus GDS(L)-lipase; enzyme inhibition; active site serine; MALDI mass spectrometry

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Podaci o prilogu

2004.

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objavljeno

Podaci o matičnoj publikaciji

Zagreb:

Podaci o skupu

Kongres Hrvatskog društva za biokemiju i molekularnu biologiju, HDBMB2004

poster

30.09.2004-02.10.2004

HOC Bjelolasica, Hrvatska

Povezanost rada

Kemija, Biologija