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Noncovalent binding of thioxylo-oligosaccharide inhibitors, methyl 4-thio-a-xylobioside (S-Xyl2-Me), methyl 4,4 II -dithio-a-xylotrioside (S-Xyl3-Me), methyl 4,4 II ,4 III -trithio-a-xylotetroside (S-Xyl4-Me), and methyl 4,4 II , 4 III ,4 IV -tetrathio-a-xylopentoside (S-Xyl5-Me), to three family 11 endo-1,4-b-xylanases from Trichoderma reesei(TRX I and TRX II) and Chaetomium thermophilum (CTX) was characterized using electrospray ionization Fourier transform ion cyclotron resonance (FT-ICR) MS and X-ray crys-tallography. . | ềFEBS Journal Determination of thioxylo-oligosaccharide binding to family 11 xylanases using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and X-ray crystallography 1 1ki- 1 2a. 3 Janne Janis Johanna Hakanpaa Nina Hakulinen Farid M. Ibatullin Antuan Hoxha Peter J. Derrick3 Juha Rouvinen1 and Pirjo Vainiotalo1 1 Department of Chemistry University of Joensuu Finland 2 Biophysics Division Petersburg Nuclear Physics Institute Gatchina Russia 3 Department of Chemistry Mass Spectrometry Institute University of Warwick Coventry UK Keywords Fourier transform ion cyclotron resonance FT-ICR noncovalent binding thioxylooligosaccharides X-ray crystallography xylanases Correspondence P. Vainiotalo Department of Chemistry University of Joensuu PO Box 111 FI-80101 Joensuu Finland Fax 358 13 2513360 Tel 358 13 2513362 E-mail pirjo.vainiotalo@joensuu.fi Received 4 January 2005 revised 1 March 2005 accepted 10 March 2005 doi 10.1111 j.1742-4658.2005.04659.x Noncovalent binding of thioxylo-oligosaccharide inhibitors methyl 4-thio-a-xylobioside S-Xyl2-Me methyl 4 4II-dithio-a-xylotrioside S-Xyl3-Me methyl 4 4II 4III-trithio-a-xylotetroside S-Xyl4-Me and methyl 4 4n 4III 4IV-tetrathio-a-xylopentoside S-Xyl5-Me to three family 11 endo-1 4-b-xylanases from Trichoderma reesei TRX I and TRX II and Chaetomium thermophilum CTX was characterized using electrospray ionization Fourier transform ion cyclotron resonance FT-ICR MS and X-ray crystallography. Ultra-high mass-resolving power and mass accuracy inherent to FT-ICR allowed mass measurements for noncovalent complexes to within DM average of 2 p.p.m. The binding constants determined by MS titration experiments were in the range 104-103 M-1 decreasing in the series of S-Xyl5-Me S-Xyl4-Me S-Xyl3-Me. In contrast S-Xyl2-Me did not bind to any xylanase at the initial concentration of 5-200 IM indicating increasing affinity with increasing number of xylopyranosyl units with a minimum requirement of three. .
