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Analysis of the dynamic properties of Bacillus circulans xylanase upon formation of a covalent glycosyl-enzyme intermediate

Published online by Cambridge University Press:  01 March 2000

GREGORY P. CONNELLY ,
STEPHEN G. WITHERS  and
LAWRENCE P. McINTOSH
GREGORY P. CONNELLY
Affiliation:
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada
STEPHEN G. WITHERS
Affiliation:
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada Department of Chemistry, The Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
LAWRENCE P. McINTOSH
Affiliation:
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada Department of Chemistry, The Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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Abstract

NMR spectroscopy was used to search for mechanistically significant differences in the local mobility of the main-chain amides of Bacillus circulans xylanase (BCX) in its native and catalytically competent covalent glycosyl-enzyme intermediate states. 15N T1, T2, and 15N{1H} NOE values were measured for ~120 out of 178 peptide groups in both the apo form of the protein and in BCX covalently modified at position Glu78 with a mechanism-based 2-deoxy-2-fluoro-β-xylobioside inactivator. Employing the model-free formalism of Lipari and Szabo, the measured relaxation parameters were used to calculate a global correlation time (τm) for the protein in each form (9.2 ± 0.2 ns for apo-BCX; 9.8 ± 0.3 ns for the modified protein), as well as individual order parameters for the main-chain NH bond vectors. Average values of the order parameters for the protein in the apo and complexed forms were S2 = 0.86 ± 0.04 and S2 = 0.91 ± 0.04, respectively. No correlation is observed between these order parameters and the secondary structure, solvent accessibility, or hydrogen bonding patterns of amides in either form of the protein. These results demonstrate that the backbone of BCX is well ordered in both states and that formation of the glycosyl-enzyme intermediate leads to little change, in any, in the dynamic properties of BCX on the time scales sampled by 15N-NMR relaxation measurements.

Keywords

glucanaseglycosyl-enzyme intermediateNMRorder parameterprotein structurerelaxation dynamics
Type
Research Article
Information
Protein Science , Volume 9 , Issue 3 , March 2000 , pp. 512 - 524
Copyright
© 2000 The Protein Society

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