Chitinase-catalyzed hydrolysis of 4-nitrophenyl penta-N-acetyl-β-chitopentaoside as determined by real-time ESIMS: The 4-nitrophenyl moiety of the substrate interacts with the enzyme binding site

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 −2 ∼ +3 and less frequently to −3 ∼ +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 −3 ∼ +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.