Kinetic evidence related to substrate-assisted catalysis of family 18 chitinases

被引:16
作者
Honda, Y [1 ]
Kitaoka, M [1 ]
Hayashi, K [1 ]
机构
[1] Natl Food Res Inst, Tsukuba, Ibaraki 3058642, Japan
来源
FEBS LETTERS | 2004年 / 567卷 / 2-3期
关键词
chitobiose phosphorylase; chitinase; glycoside hydrolase family 18; 4-methylumbelliferyl chitobioside; substrate-assisted catalysis; Serratia marcescens;
D O I
10.1016/j.febslet.2004.05.002
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The hydrolytic reaction of family 18 chitinase has been considered to occur via substrate assisted catalysis. To kinetically investigate the enzyme reaction mechanism, we synthesized compounds designed to reduce the polarization of the carbonyl in N acetyl group, GlcNAc-GlcN(TFA)-UMB (2) and GlcNAc-GlcN(TAc)-UMB (3). Kinetic parameters in the hydrolysis of these compounds by chitinase A from Serratia marcescens (ChiA) were compared with those from the hydrolysis of (GlcNAC)(2)-UMB (1). The k(cat) of 2 was 3.4% of 1, but the K-m of 2 was 10-fold that of 1. In contrast, the k(cat) of 3 was only 0.3%, of that of 1, and the two reactions had an identical K-m. The drastic decreases in k(cat) were probably due to the weak nucleophilic activity of the C2-N-trifluoroacetamide and N-thioacetamide groups at reducing ends of compounds 2 and 3, respectively. These results indicate that the anchimeric assistance of the C2 N-acetamide group at GlcNAc plays a key role in the hydrolytic reactions catalyzed by family 18 chitinases. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
引用
收藏
页码:307 / 310
页数:4
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