Kinetic analysis of the reaction catalyzed by chitinase A1 from Bacillus circulans WL-12 toward the novel substrates, partially N-deacetylated 4-methylumbelliferyl chitobiosides

Yuji Honda, Shinji Tanimori, Mitsunori Kirihata, Satoshi Kaneko, Ken Tokuyasu, Masayuki Hashimoto, Takeshi Watanabe, Tamo Fukamizo

Research output: Contribution to journalArticle

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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 languageEnglish
Pages (from-to)194-197
Number of pages4
JournalFEBS Letters
Volume476
Issue number3
DOIs
Publication statusPublished - 2000 Jul 7

Fingerprint

Chitinases
Bacilli
Bacillus
Kinetics
Substrates
Fluorescent Dyes
Sugars
Catalysis
Catalyst supports
N,N-diacetylchitobiose

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Honda, Yuji ; Tanimori, Shinji ; Kirihata, Mitsunori ; Kaneko, Satoshi ; Tokuyasu, Ken ; Hashimoto, Masayuki ; Watanabe, Takeshi ; Fukamizo, Tamo. / Kinetic analysis of the reaction catalyzed by chitinase A1 from Bacillus circulans WL-12 toward the novel substrates, partially N-deacetylated 4-methylumbelliferyl chitobiosides. In: FEBS Letters. 2000 ; Vol. 476, No. 3. pp. 194-197.
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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.",
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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 journalArticle

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AU - Honda, Yuji

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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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