Kinetic analysis of the reaction catalyzed by chitinase A1 from Bacillus circulans WL-12 toward the novel substrates, partially N-deacetylated 4-methylumbelliferyl chitobiosides.
The kinetic behavior of chitinase A1 from Bacillus circulans WL-12 was investigated using the novel fluorogenic substrates, N-deacetylated 4-methylumbelliferyl chitobiosides [GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)(2)-UMB (4)], and the results were compared with those obtained using 4-methylumbelliferyl N, N'-diacetylchitobiose [(GlcNAc)(2)-UMB (1)] as the substrate. The chitinase did not release the UMB moiety from compound 4, but successfully released UMB from the other substrates. k(cat)/K(m) values determined from the releasing rate of the UMB moiety were: 145.3 for 1, 8.3 for 2, and 0.1 s(-1) M(-1) for 3. The lack of an N-acetyl group at subsite (-1) reduced the activity to a level 0.1% of that obtained with compound 1, while the absence of the N-acetyl group at subsite (-2) reduced the relative activity to 5.7%. These observations strongly support the theory that chitinase A1 catalysis occurs via a 'substrate-assisted' mechanism. Using these novel fluorogenic substrates, we were able to quantitatively evaluate the recognition specificity of subsite (-2) toward the N-acetyl group of the substrate sugar residue. The (-2) subsite of chitinase A1 was found to specifically recognize an N-acetylated sugar residue, but this specificity was not as strict as that found in subsite (-1).
Laboratory of Bioorganic Chemistry, College of Agriculture, Osaka Prefecture University, Sakai, Osaka, Japan. hondada@nfri.affrc.go.jp
Y
Y
Honda
S
S
Tanimori
M
M
Kirihata
S
S
Kaneko
K
K
Tokuyasu
M
M
Hashimoto
T
T
Watanabe
T
T
Fukamizo
D000107:Acetylation; D001407:Bacillus / Q000201:enzymology*; D002688:Chitinases / Q000378:metabolism*; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism*; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D013379:Substrate Specificity
D004187:Disaccharides; D006923:Hymecromone; D002688:Chitinases