Thermoanaerobacterium saccharolyticum β-xylosidase is a member of family 39 of the glycosyl hydrolases. This grouping comprises both retaining β-d-xylosidases and α-l-iduronidases. T. saccharolyticum β-xylosidase catalyses the hydrolysis of short xylo-oligosaccharides into free xylose via a covalent xylosyl–enzyme intermediate. Incubation of T. saccharolyticum β-xylosidase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-d-xyloside resulted in time-dependent inactivation of the enzyme (inactivation rate constant ki = 0.089 min-1, dissociation constant for the inactivator Ki = 65 µM) through the accumulation of a covalent 2-deoxy-2-fluoro-α-d-xylosyl–enzyme, as observed by electrospray MS. Removal of excess inactivator and regeneration of the free enzyme through transglycosylation with either xylobiose or thiobenzyl xyloside demonstrated that the covalent intermediate was kinetically competent. Peptic digestion of the 2-deoxy-2-fluoro-α-d-xylosyl–enzyme intermediate and subsequent analysis by electrospray ionization triple-quadrupole MS in the neutral-loss mode indicated the presence of a 2-deoxy-2-fluoro-α-d-xylosyl peptide. Sequence determination of the labelled peptide by tandem MS in the daughter-ion scan mode permitted the identification of Glu-277 (bold and underlined) as the catalytic nucleophile within the sequence IILNSHFPNLPFHITEY.
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October 1998
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Research Article|
October 15 1998
Identification of Glu-277 as the catalytic nucleophile of Thermoanaerobacterium saccharolyticum β-xylosidase using electrospray MS
David J. VOCADLO;
David J. VOCADLO
*Protein Engineering Network of Centres of Excellence of Canada and Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
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Lloyd F. MACKENZIE;
Lloyd F. MACKENZIE
*Protein Engineering Network of Centres of Excellence of Canada and Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
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Shouming HE;
Shouming HE
*Protein Engineering Network of Centres of Excellence of Canada and Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
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Gregory J. ZEIKUS;
Gregory J. ZEIKUS
†Department of Biochemistry, Michigan State University, East Lansing, MI 48824, U.S.A.
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Stephen G. WITHERS
Stephen G. WITHERS
1
*Protein Engineering Network of Centres of Excellence of Canada and Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
1To whom correspondence should be addressed (withers@chem.ubc.ca).
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Publisher: Portland Press Ltd
Received:
March 20 1998
Revision Received:
July 10 1998
Accepted:
August 11 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 335 (2): 449–455.
Article history
Received:
March 20 1998
Revision Received:
July 10 1998
Accepted:
August 11 1998
Citation
David J. VOCADLO, Lloyd F. MACKENZIE, Shouming HE, Gregory J. ZEIKUS, Stephen G. WITHERS; Identification of Glu-277 as the catalytic nucleophile of Thermoanaerobacterium saccharolyticum β-xylosidase using electrospray MS. Biochem J 15 October 1998; 335 (2): 449–455. doi: https://doi.org/10.1042/bj3350449
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