Kinetic Analysis of Barley Chitinase

The endochitinase from barley is the archetypal enzyme for a large class of plant-derived antifungal chitinases. The X-ray structure was solved previously in our laboratory and a mechanism of action proposed based on structural considerations. In this manuscript we report the use of a defined soluble substrate, 4-methylumbelliferyl β-N,N′,N″-triacetylchitotrioside, to characterize kinetic parameters of the enzyme. The pH profile shows that activity is controlled by a base with a pKaof 3.9 (Glu 89) and an acid with a pKaof 6.9 (Glu 67). TheKmusing the synthetic substrate is 33 μm, and thekcatis 0.33 min−1, while theKmfor (GlcNAc)4is 3 μmandkcatis 35 min−1. Binding constants were measured for β-linked oligomers ofN-acetylglucosamine. The monomer does not bind and dissociation constants for the dimer, trimer, and tetramer are 43, 19, and 6 μm, respectively. Analysis of kinetic and dissociation constants proves the mechanism of barley chitinase is consistent with a Bi–Bi kinetic model for hydrolysis, with (GlcNAc)4and water as substrates and (GlcNAc)2as products. Substrate cleavage patterns show that (GlcNAc)6is cleaved in half to (GlcNAc)3as well as into (GlcNAc)4and (GlcNAc)2with almost equal efficiency. NMR analysis of cleavage products confirms that the enzyme proceeds with anomeric inversion of products.