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Characterization of covalently inhibited extracellular lipase from Streptomyces rimosus by matrix‐assisted laser desorption/ionization time‐of‐flight and matrix‐assisted laser desorption/ionization quadrupole ion trap reflectron time‐of‐flight mass spectrometry: localization of the active site serine
Author(s) -
Zehl Martin,
Leščić Ivana,
Abramić Marija,
Rizzi Andreas,
KojićProdić Biserka,
Allmaier Günter
Publication year - 2004
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.750
Subject(s) - chemistry , matrix assisted laser desorption/ionization , mass spectrometry , lipase , chromatography , electrospray ionization , serine , desorption , enzyme , biochemistry , organic chemistry , adsorption
A chemical modification approach combined with matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry was used to identify the active site serine residue of an extracellular lipase from Streptomyces rimosus R6‐554W. The lipase, purified from a high‐level overexpressing strain, was covalently modified by incubation with 3,4‐dichloroisocoumarin, a general mechanism‐based serine protease inhibitor. MALDI time‐of‐flight (TOF) mass spectrometry was used to probe the nature of the intact inhibitor‐modified lipase and to clarify the mechanism of lipase inhibition by 3,4‐dichloroisocoumarin. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound to the lipase. The MALDI matrix 2,6‐dihydroxyacetophenone facilitated the formation of highly abundant [M + 2H] 2+ ions with good resolution compared to other matrices in a linear TOF instrument. This allowed the detection of two different inhibitor‐modified lipase species. Exact localization of the modified amino acid residue was accomplished by tryptic digestion followed by low‐energy collision‐induced dissociation peptide sequencing of the detected 2‐(carboxychloromethyl)benzoylated peptide by means of a MALDI quadrupole ion trap reflectron TOF instrument. The high sequence coverage obtained by this approach allowed the confirmation of the site specificity of the inhibition reaction and the unambiguous identification of the serine at position 10 as the nucleophilic amino acid residue in the active site of the enzyme. This result is in agreement with the previously obtained data from multiple sequence alignment of S. rimosus lipase with different esterases, which indicated that this enzyme exhibits a characteristic Gly‐Asp‐Ser‐(Leu) motif located close to the N‐terminus and is harboring the catalytically active serine residue. Therefore, this study experimentally proves the classification of the S. rimosus lipase as GDS(L) lipolytic enzyme. Copyright © 2004 John Wiley & Sons, Ltd.