Abstract
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). Copyright (C) 2000 Federation of European Biochemical Societies.
| Original language | English |
|---|---|
| Pages (from-to) | 194-197 |
| Number of pages | 4 |
| Journal | FEBS Letters |
| Volume | 476 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2000 Jul 7 |
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All Science Journal Classification (ASJC) codes
- Biophysics
- Structural Biology
- Biochemistry
- Molecular Biology
- Genetics
- Cell Biology
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Kinetic analysis of the reaction catalyzed by chitinase A1 from Bacillus circulans WL-12 toward the novel substrates, partially N-deacetylated 4-methylumbelliferyl chitobiosides. / Honda, Yuji; Tanimori, Shinji; Kirihata, Mitsunori; Kaneko, Satoshi; Tokuyasu, Ken; Hashimoto, Masayuki; Watanabe, Takeshi; Fukamizo, Tamo.
In: FEBS Letters, Vol. 476, No. 3, 07.07.2000, p. 194-197.Research output: Contribution to journal › Article
TY - JOUR
T1 - Kinetic analysis of the reaction catalyzed by chitinase A1 from Bacillus circulans WL-12 toward the novel substrates, partially N-deacetylated 4-methylumbelliferyl chitobiosides
AU - Honda, Yuji
AU - Tanimori, Shinji
AU - Kirihata, Mitsunori
AU - Kaneko, Satoshi
AU - Tokuyasu, Ken
AU - Hashimoto, Masayuki
AU - Watanabe, Takeshi
AU - Fukamizo, Tamo
PY - 2000/7/7
Y1 - 2000/7/7
N2 - 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). Copyright (C) 2000 Federation of European Biochemical Societies.
AB - 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). Copyright (C) 2000 Federation of European Biochemical Societies.
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U2 - 10.1016/S0014-5793(00)01729-4
DO - 10.1016/S0014-5793(00)01729-4
M3 - Article
VL - 476
SP - 194
EP - 197
JO - FEBS Letters
JF - FEBS Letters
SN - 0014-5793
IS - 3
ER -