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

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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).

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