The role of tryptophan residues in substrate binding to catalytic domains A and B of xylanase C from Fibrobacter succinogenes S85.
Oxidation of the isolated catalytic domain B of xylanase C (XynC-B) from Fibrobacter succinogenes with N-bromosuccinimide (NBS) resulted in the modification of five of the seven Trp residues present in the enzyme. Hydrolytic activity of the enzyme was rapidly lost upon initiation of oxidation as a molar ratio of about two NBS molecules per molar equivalent of protein was sufficient to cause 50% inhibition of enzyme activity, and the addition of five molar equivalents of NBS resulted in less than 10% activity. Pre-incubation of XynC-B with the competitive inhibitor D-xylose resulted in the apparent protection of two Trp residues from oxidation. Xylose protection of the enzyme also resulted in a maintenance of activity, with 60% activity still evident after addition of 8-9 molar equivalents of NBS. This protection from inactivation was enhanced by the inclusion of xylohexaose in reaction mixtures. Under these conditions, however, a further Trp residue was protected from NBS oxidation. The three protected Trp residues were identified as Trp135, Trp161 and Trp202 by differential labelling and peptide mapping of NBS-oxidized preparations of the xylanase employing a combination of electrospray mass spectroscopic analysis and N-terminal sequencing. By analogy to the known structures of the family 11 xylanases, the fully conserved Trp202 residue is located on the only alpha-helix present in the enzymes, at the interface between it and the back of the beta-sheet which forms the active site cleft. Trp135 represents a highly conserved aromatic residue in family 11, but it is replaced with Thr in domain A of F. succinogenes xylanase C. To investigate the role of Trp135 in conferring the different activity profile of domain B relative to domain A, the Trp135Thr and Trp135Ala derivatives of domain B were prepared by site-directed mutagenesis. However, the kinetic parameters of the two domain B derivatives were not significantly different compared to the wild-type enzyme as reflected by K(M) and k(cat) values and product distribution profiles. Similar results were obtained with the Trp161Ala derivative of domain B, indicating that these two residues do not directly participate in the binding of substrate but likely form the foundation for binding subsite 2.
McAllister
K A
KA
Department of Microbiology, University of Guelph, Ont., N1G 2W1, Guelph, Canada.
Marrone
L
L
Clarke
A J
AJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D043364:Endo-1,4-beta Xylanases; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014364:Tryptophan / Q000378:metabolism*; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D014364:Tryptophan; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases