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 beta-N,N',N"-triacetylchitotrioside, to characterize kinetic parameters of the enzyme. The pH profile shows that activity is controlled by a base with a pKa of 3.9 (Glu 89) and an acid with a pKa of 6.9 (Glu 67). The Km using the synthetic substrate is 33 microM, and the k(cat) is 0.33 min(-1), while the Km for (GlcNAc)4 is 3 microM and k(cat) is 35 min(-1). Binding constants were measured for beta-linked oligomers of N-acetylglucosamine. The monomer does not bind and dissociation constants for the dimer, trimer, and tetramer are 43, 19, and 6 microM, 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)4 and water as substrates and (GlcNAc)2 as products. Substrate cleavage patterns show that (GlcNAc)6 is cleaved in half to (GlcNAc)3 as well as into (GlcNAc)4 and (GlcNAc)2 with almost equal efficiency. NMR analysis of cleavage products confirms that the enzyme proceeds with anomeric inversion of products.
Hollis
T
T
Department of Chemistry and Biochemistry, University of Texas, Austin 78712, USA.
Honda
Y
Y
Fukamizo
T
T
Marcotte
E
E
Day
P J
PJ
Robertus
J D
JD
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D002240:Carbohydrate Sequence; D002686:Chitin / Q000031:analogs & derivatives / Q000378:metabolism; D002688:Chitinases / Q000378:metabolism*; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D014312:Trisaccharides / Q000378:metabolism
D005456:Fluorescent Dyes; D009844:Oligosaccharides; D014312:Trisaccharides; D002686:Chitin; C003387:tetra-N-acetylchitotetraose; C019117:N,N',N''-triacetylchitotriose; D002688:Chitinases; D000117:Acetylglucosamine