Kinetic evidence related to substrate-assisted catalysis of family 18 chitinases.
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 kcat of 2 was 3.4% of 1, but the Km of 2 was 10-fold that of 1. In contrast, the kcat of 3 was only 0.3% of that of 1, and the two reactions had an identical Km. The drastic decreases in kcat 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.
Honda
Yuji
Y
National Food Research Institute, 2-1-12, Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
Kitaoka
Motomitsu
M
Hayashi
Kiyoshi
K
D000107:Acetylation; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007700:Kinetics; D012706:Serratia marcescens / Q000201:enzymology; D013379:Substrate Specificity; D014468:Umbelliferones / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D004187:Disaccharides; D005456:Fluorescent Dyes; D014468:Umbelliferones; C032438:chitobiose; D002688:Chitinases