Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10605599 | Carbohydrate Research | 2011 | 4 Pages |
Abstract
4-Nitrophenyl penta-N-acetyl-β-chitopentaoside [(GlcNAc)5-pNP] was hydrolyzed by a family GH-19 class II barley chitinase, and the enzymatic reaction was monitored by real-time ESIMS. The wild-type enzyme hydrolyzed (GlcNAc)5-pNP producing predominantly (GlcNAc)3-pNP and a lesser amount of (GlcNAc)2-pNP, indicating that the (GlcNAc)5 portion of the substrate binds predominantly to subsites â2 â¼Â +3 and less frequently to â3 â¼Â +2. However, (GlcNAc)2-pNP was mainly produced from (GlcNAc)5-pNP by mutated enzymes, in which Trp72 and Trp82 located at +3/+4 were substituted with alanine (W72A and W72A/W82A), indicating that the (GlcNAc)5 portion of the substrate binds predominantly to subsites â3 â¼Â +2 of the mutants. The mutations of the tryptophan residues resulted in a significant shift of the substrate-binding mode to the glycon side, supporting the idea that the indole side chain of Trp72 interacts with the 4-nitrophenyl moiety of the substrate at subsite +4.
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Physical Sciences and Engineering
Chemistry
Organic Chemistry
Authors
Thomas Letzel, Ellen Sahmel-Schneider, Karen Skriver, Takayuki Ohnuma, Tamo Fukamizo,