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Kinetic evidence related to substrate‐assisted catalysis of family 18 chitinases
Author(s) -
Honda Yuji,
Kitaoka Motomitsu,
Hayashi Kiyoshi
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2004.05.002
Subject(s) - enzyme kinetics , chemistry , hydrolysis , acetamide , serratia marcescens , chitinase , catalysis , substrate (aquarium) , nucleophile , medicinal chemistry , stereochemistry , enzyme , organic chemistry , active site , biochemistry , oceanography , escherichia coli , gene , geology
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.