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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

2000; Wiley; Volume: 476; Issue: 3 Linguagem: Inglês

10.1016/s0014-5793(00)01729-4

1873-3468

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

Legume Nitrogen Fixing Symbiosis

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).