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Lookup NU author(s): Simon Andrews,
Emeritus Professor Harry Gilbert
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Pseudomonas cellulosa xylanase 10A (Pc Xyn10A) contains an extended substrate binding cleft comprising three glycone (-1 to -3) and four aglycone (+1 to +4) subsites and, typical of retaining glycoside hydrolases, exhibits transglycosylation activity at elevated substrate concentrations. In a previous study [Charnock, S. J., et al. (1997) J. Biol. Chem. 272, 2942-2951], it was demonstrated that the -2 subsite mutations E43A and N44A caused a 100-fold reduction in activity against xylooligosaccharides, but did not influence xylanase activity. This led to the proposal that the low activity of these mutants against xylooligosaccharides was due to nonproductive complex formation between these small substrates and the extended aglycone region of the active site. To test this hypothesis, key residues at the +2 (Asn182), +3 (Tyr255), and +4 (Tyr220) subsites were substituted for alanine, and the activity of the mutants against polysaccharides and oligosaccharides was evaluated. All the aglycone mutants exhibited greatly reduced or no transglycosylating activity, and the triple mutants, E43A/Y220A/Y255A and E43A/N182A/Y255A, had activity against xylotriose similar to that of E43A. The aglycone mutations caused an increase in both kcat and Km against xylan, with N182A/Y220A/Y255A and N182A/Y255A exhibiting 25- and 15-fold higher kcat values, respectively, than wild-type Pc Xyn10A. These data indicate that Glu43 plays a role in binding xylooligosaccharides, but not xylan, suggesting that the mechanisms by which Pc Xyn10A binds polysaccharides and oligosaccharides are distinct. The increased kcat of the mutants against xylan indicates that the aglycone region of wild-type Pc Xyn10A restricts the rate of catalysis by limiting diffusion of the cleaved substrate, generated at the completion of the k2 step, out of the active site.
Author(s): Armand S, Andrews SR, Charnock SJ, Gilbert HJ
Publication type: Article
Publication status: Published
ISSN (print): 0006-2960
ISSN (electronic): 1520-4995
Publisher: American Chemical Society
PubMed id: 11412093
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