Influence of the aglycone region of the substrate binding cleft of Pseudomonas xylanase 10A on catalysis

Pseudomonas cellulosa xylanase 10A (Pc Xyn10A) contains an extended substra te binding cleft comprising three,glycone (-1 to -3) and four aglycone (+ 1 to +4) subsites and, typical of retaining glycoside hydrolases, exhibits t ransglycosylation activity at elevated substrate concentrations. In a previ ous study [Charnock, S. J., et al. (1997) J. Biol. Chem. 272, 2942-2951], i t was demonstrated that the -2 subsite mutations E43A and N44A caused a 100 -fold reduction in activity against xylooligosaccharides, but did not influ ence xylanase activity. This led to the proposal that the low activity of t hese mutants against xylooligosaccharides was due to nonproductive complex formation between these small substrates and the extended aglycone region o f the active site. To test this hypothesis, key residues at the +2 (Asn182) , +3 (Tyr255), and +4 (Tyr220) subsites were substituted for alanine, and t he activity of the mutants against polysaccharides and oligosaccharides was evaluated. All the aglycone mutants exhibited greatly reduced or no transg lycosylating activity, and the triple mutants, E43A/Y220A/Y255A and E43A/N1 82A/ Y255A, had activity against xylotriose similar to that of E43A. The ag lycone mutations caused an increase in both k(cat) and K-m against xylan, w ith N182A/Y220A/Y255A and N182A/Y255A exhibiting 25- and 15-fold higher k(c at) values, respectively, than wild-type Pc Xyn10A. These data indicate tha t Glu43 plays a role in binding xylooligosaccharides, but not xylan, sugges ting that the mechanisms by which Pc Xyn10A binds polysaccharides and oligo saccharides are distinct. The increased k(cat) of the mutants against xylan indicates that the aglycone region of wild-type Pc Xyn10A restricts the ra te of catalysis by Limiting diffusion of the cleaved substrate, generated a t the completion of the k(2) step, out of the active site.