Abstract
Kinetic analysis was done on chitinase E3 (EC 3.2.1.14) from yam, Dioscorea opposita THUNB, using both series of N-acetylchitooligosaccharides (GlcNAcn, n=2 to 6) and p-nitrophenyl N-acetylchitooligosaccharides (pNp-GlcNAcn, n=1 to 5) as substrates. The enzyme cleaved GlcNAc3 to GlcNAc plus GlcNAc2, GlcNAc4 to two molecules of GlcNAc2, GlcNAc5 to GlcNAc2 plus GlcNAc3, and GlcNAc6 by three ways to GlcNAc plus GlcNAc5 (32%), GlcNAc2 plus GlcNAc4 (42%) and two molecules of GlcNAc3 (26%). The speed of the reaction was observed in the following order, GlcNAc4>GlcNAc5>GlcNAc6>GlcNAc3. Stronger substrate inhibition was observed in the longer chain substrates. The reactions of pNp-GlcNAcn (n=1 to 5) were similar to those of GlcNAcn (n=2 to 6), respectively. The cleavage sites of pNp-GlcNAc2 and pNp-GlcNAc3 were the second β-1, 4-linkages from the reducing end side, and the main cleavage sites of pNp-GlcNAc4 and pNp-GlcNAc5 were the third linkages. p-Nitrophenol was not released from all p-nitrophenyl derivatives. The speed of the reaction was observed in the following order, pNp-GlcNAc4 > pNp-GlcNAc5 > pNp-GlcNAc3 > pNp-GlcNAc2. Neither GlcNAc2 nor pNp-GlcN Ac was cleaved. These results suggest that yam chitinase E3 acts in a random fashion.
