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

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

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Skup
Kongres Hrvatskog društva za biokemiju i molekularnu biologiju, HDBMB2004

Mjesto i datum
HOC Bjelolasica, Hrvatska, 30.09.2004. - 02.10.2004

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

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

Sažetak
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.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA