Kinetic and molecular properties of Bacillus subtilis IBTC-3 subtilisin.
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.
Głowacka
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Institute of Technical Biochemistry, Technical University of Lodz, Stefanowskiego 4/10 Str., 90-924 Lodz, Poland.
Podstawka
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Szczesna-Antczak
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Kalinowska
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Antczak
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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*
D013381:Subtilisins; C502515:IBTC-3 subtilisin, Bacillus subtilis; D020860:Subtilisin