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.