title: Kinetic and molecular properties of Bacillus subtilis IBTC-3 subtilisin.
abstract: Bacillus subtilis IBTC-3 subtilisin was purified by gel filtration on Sephadex G 75 and affinity chromatography on bacitracin-CNBr-Sepharose 4B and characterized. Its molecular mass of 27 kDa was determined by SDS-PAGE, and isoelectric pH of 8.4 by chromatofocusing. FT-Raman and FT-IR spectroscopy studies revealed fragments with alpha-helix and irregular secondary structures within the polypeptide chain. The beta-sheet conformation was observed only in second-derivatives of FT-RS and FT-IR spectra, in the range of the amide II, III, and I bands. Tyr residues were shown to be hydrogen bonded and CSCH(3) groups adopted two conformations (P(H)-T and P(C)-G conformers). Kinetic properties of B. subtilis IBTC-3 subtilisin in hydrolysis of ethyl esters of amino acid derivatives were compared with that of alkaline peptidase from Bacillus alcalophilus PB92. The first enzyme displayed the highest affinity for NAc-Phe-OEt, both in hydrolysis (K(m) of 0.22 mM) and in synthesis (K(m) of 0.85 mM), whereas PB92 peptidase preferred Tyr derivatives (NAc-Tyr-OEt, K(m) of 0.043 and 0.75 mM, respectively). In contrast to the latter enzyme, B. subtilis IBTC-3 subtilisin catalyzed hydrolysis and synthesis of Bz-Arg-OEt.
authors.affiliation: Institute of Technical Biochemistry, Technical University of Lodz, Stefanowskiego 4/10 Str., 90-924 Lodz, Poland.
authors.forename: A E
authors.initials: AE
authors.lastname: Głowacka
authors.forename: E
authors.initials: E
authors.lastname: Podstawka
authors.forename: M H
authors.initials: MH
authors.lastname: Szczesna-Antczak
authors.forename: H
authors.initials: H
authors.lastname: Kalinowska
authors.forename: T
authors.initials: T
authors.lastname: Antczak
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D020860:Subtilisin / Q000737:chemistry* / Q000378:metabolism*; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D013381:Subtilisins; C502515:IBTC-3 subtilisin, Bacillus subtilis; D020860:Subtilisin