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

AbstractThe 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. kcat/Km 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)