| Autor: |
Leščić, Ivana, Kovačić, Filip, Zehl, Martin, Abramić, Marija, Allmaier, Günter, Luić, Marija, Kojić-Prodić, Biserka |
| Jazyk: |
angličtina |
| Rok vydání: |
2004 |
| Předmět: |
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| Popis: |
A novel GDS(L) lipase (EC 3.1.1.3) from Streptomyces rimosus R6-554W was purified and biochemically characterized ; cloning and high level expression of its gene were described and sequence was deduced by bioinformatic approach. The mass spectrometry with MALDI-TOF and MALDI-QIT-RTOF were used to characterize wild-type and overexpressed lipase (SrL). The molecular mass was determined, 24165.76 Da. The intramolecular disulfide bonds C27-C52, C93-C101 and C151-C198 were determined. So far crystallization experiments have not provided good quality crystals for diffraction experiments. To stabilize SrL a few complexes with the inhibitors were prepared and crystallization experiments are in due course. In order to examine the inhibitor activity of our novel lipase we tested 3, 4-dichloro- isocoumarin (DCI) - typical inhibitor for serine proteases, and tetrahydrolipstatin (THL) - a pancreatic lipase inhibitor ; the experiments with 1-O-hexadecyl-2-O-perylenedodecyl-sn-glycero-3-phosphonic acid-(n-hexyl)-p-nitrophenyl-ester - a characteristic lipase inhibitor are initiated. Incubation of SrL with different concentrations of phenylmethylsulfonyl fluoride (PMSF) has not decreased 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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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