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

TY - JOUR

T1 - Chitinase-catalyzed hydrolysis of 4-nitrophenyl penta-N-acetyl-B-chitopentaoside as determined by real-time ESI-MS

T2 - The 4-nitrophenyl moiety of the substrate interacts with the enzyme binding site

AU - Letzel, T.

AU - Sahmel-Schneide, E.

AU - Skriver, K.

AU - Ohnuma, T.

AU - Fukamizo, T.

PY - 2011/5/1

Y1 - 2011/5/1

N2 - 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.

AB - 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.

U2 - 10.1016/j.carres.2011.01.012

DO - 10.1016/j.carres.2011.01.012

M3 - Journal article

C2 - 21397215

SN - 0008-6215

VL - 346

SP - 863

EP - 866

JO - Carbohydrate Research

JF - Carbohydrate Research

IS - 6

ER -