poly[1,4-(N-acetyl-beta-D-glucosaminide)] glycanohydrolase
NAG-NAG-NAG-NAG-NAG (chain B), NAG-NAG (chain C)
REVISIONS, AND X-RAY CRYSTALLOGRAPHY (2.3 ANGSTROMS).
Crystal structure of a bacterial chitinase at 2.3 A resolution.
The 1.8 A resolution structure of hevamine, a plant chitinase/lysozyme, and analysis of the conserved sequence and structure motifs of glycosyl hydrolase family 18.
Evolution of immunoglobulin-like modules in chitinases: their structural flexibility and functional implications.
Site-directed mutagenesis of Asp313, Glu315, and Asp391 residues in chitinase of Aeromonas caviae.
Structure of a two-domain chitotriosidase from Serratia marcescens at 1.9-A resolution.
High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis.
Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases.
De novo purification scheme and crystallization conditions yield high-resolution structures of chitinase A and its complex with the inhibitor allosamidin.
Family 18 chitinase-oligosaccharide substrate interaction: subsite preference and anomer selectivity of Serratia marcescens chitinase A.
(5) The deprotonated sidechain of Glu315 acts as a general base, abstracting the water molecule, which can act on the C1 carbon at subsite (-1). Taken together, Glu315 acts as an acid-base, whilst Asp313 modulates the role of Glu315. Moreover, Tyr390 stabilizes the transition-state through the water. The stabilization might be assisted by N-acetyl group of the substrate. This enzyme is composed of the N-terminal immunoglobulin-like region and the C-terminal catalytic region, which has also two distinct domains. According to the literature [3], the N-terminal immunoglobulin-like domain might interact with the chitin substrate during catalysis. In the early study [4], Asp313, Glu315 and Asp391 have been thought to be involved in catalysis. The paper [4] suggested that Asp313 and Glu315 might act as a stabilizer and a general acid, respectively. According to the more recent work ([6] & [8]), however, Tyr390 was reported to be involved in the catalytic reaction, instead of Asp391. The paper [6] proposed the following mechanism: (1) The sugar unit at the subsite (-1) adopts a boat conformation, which has higher free energy by 8kcal/mol, and its N-acetyl group points toward Asp313 and Glu315. The side chain of Asp313 points toward Asp311, away from Glu315. A water molecule is hydrogen-bonded to the phenol hydroxyl group of Tyr390 and the amine group of the acetamido group of the sugar unit at the subsite (-1). The sidechain carboxylate of Glu315 is protonated. (2) Glu315 functions as a general acid, and protonates the O4 oxygen of the sugar unit at the subsite (+1), thus cleaving the glycosidic linkage, O4(+1)-C1(-1). (This suggests SN1-like cleavage.) (3) The positive charge is developed on C1 and O5 atoms at the subsite (-1), which can be stabilized by the water molecule hydrogen-bonded to Tyr390 and the acetamido group at (-1), the O7 atom of the acetamido group at (-1), and the deprotonated sidechain of Glu315. (4) This cleavage induces the rotation of Asp313 toward Glu315, and the rotation of the acetamido group at (-1) toward Tyr390. The rotation of the acetamido group translocates the water molecule bound to Tyr390 closer to Glu315. However, the mutation of Asp391 inactivates this enzyme, suggesting that the residue must be involved in catalysis (see Table 1 of [6]).