KINETIC-ANALYSIS OF BARLEY CHITINASE

The endochitinase from barley is the archetypal enzyme for a large cla ss of plant-derived antifungal chitinases. The X-ray structure was sol ved previously in our laboratory and a mechanism of action proposed ba sed on structural considerations. In this manuscript we report the use of a defined soluble substrate, 4-methylumbelliferyl beta-N,N',N ''-t riacetylchitotrioside, to characterize kinetic parameters of the enzym e. The pH profile shows that activity is controlled by a base with a p K(a) of 3.9 (Glu 89) and an acid with a pK(a) of 6.9 (Glu 67). The K-m using the synthetic substrate is 33 mu M, and the k(cat) is 0.33 min( -1), while the K-m for (GlcNAc)(4) is 3 mu M and k(cat) is 35 min(-1). Binding constants were measured for beta-linked oligomers of N-acetyl glucosamine. The monomer does not bind and dissociation constants for the dimer, trimer, and tetramer are 43, 19, and 6 mu M, respectively. Analysis of kinetic and dissociation constants proves the mechanism of barley chitinase is consistent with a Bi-Bi kinetic model for hydroly sis, with (GlcNAc)(4) and water as substrates and (GlcNAc)(2) as produ cts. Substrate cleavage patterns show that (GlcNAc)(6) is cleaved in h alf to (GlcNAc)(3) as well as into (GlcNAc)(4) and (GlcNAc)(2) with al most equal efficiency. NMR analysis of cleavage products confirms that the enzyme proceeds with anomeric inversion of products. (C) 1997 Aca demic Press.