Influence of the Aglycone Region of the Substrate Binding Cleft of Pseudomonas Xylanase 10A on Catalysis

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• 作者：Sylvie Armand ; Simon R. Andrews ; Simon J. Charnock ; and Harry J. Gilbert
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：June 26, 2001
• 年：2001
• 卷：40
• 期：25
• 页码：7404 - 7409
• 全文大小：90K
• 年卷期：v.40,no.25(June 26, 2001)
• ISSN：1520-4995

文摘

Pseudomonas cellulosa xylanase 10A (Pc Xyn10A) contains an extended substrate bindingcleft comprising three glycone (-1 to -3) and four aglycone (+1 to +4) subsites and, typical of retainingglycoside hydrolases, exhibits transglycosylation activity at elevated substrate concentrations. In a previousstudy [Charnock, S. J., et al. (1997) J. Biol. Chem. 272, 2942-2951], it was demonstrated that the -2subsite 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 mutantsagainst xylooligosaccharides was due to nonproductive complex formation between these small substratesand 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 mutantsagainst polysaccharides and oligosaccharides was evaluated. All the aglycone mutants exhibited greatlyreduced 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 increasein both k_cat and K_m against xylan, with N182A/Y220A/Y255A and N182A/Y255A exhibiting 25- and15-fold higher k_cat values, respectively, than wild-type Pc Xyn10A. These data indicate that Glu43 playsa role in binding xylooligosaccharides, but not xylan, suggesting that the mechanisms by which Pc Xyn10Abinds polysaccharides and oligosaccharides are distinct. The increased k_cat of the mutants against xylanindicates that the aglycone region of wild-type Pc Xyn10A restricts the rate of catalysis by limiting diffusionof the cleaved substrate, generated at the completion of the k₂ step, out of the active site.