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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
Author(s) -
Honda Yuji,
Tanimori Shinji,
Kirihata Mitsunori,
Kaneko Satoshi,
Tokuyasu Ken,
Hashimoto Masayuki,
Watanabe Takeshi,
Fukamizo Tamo
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01729-4
Subject(s) - bacillus circulans , chitinase , chemistry , moiety , residue (chemistry) , substrate (aquarium) , stereochemistry , catalysis , enzyme , organic chemistry , biology , ecology
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).