Subsite structure of the endo-type chitin deacetylase from a Deuteromycete, Colletotrichum lindemuthianum:: an investigation using steady-state kinetic analysis and MS

The endo-type chitin deacetylase (EC 3.5.1.41) from a Deuteromycete, Colletotrichum lindemuthianum (ATCC 56676), catalyses the hydrolysis of the acetamido group of GlcNAc (2-acetamido-2-deoxy-D-glucose) residues in chitin or chito-oligosaccharides with a degree of polymerization (n) equal to or g-state kinetic parameters for the initial greater than 2. The steady deacetylation reactions of (GlcNAc)(2-6) were determined using a direct, continuous spectrophotometric assay in combination with ESI-MS (electrospray ionization MS) analysis of the products. The dependence of the observed K-m and k(cat)/K-m on n suggests the presence of four enzyme subsites (-2, -1, 0 and +1) that interact with GlcNAc residues from the non-reducing end to the reducing end of the substrate. The turnover number (k(cat), 7 s(-1)) is independent of it and represents the intrinsic rate constant (k(int)) for the hydrolysis of the acetamido group in subsite 0. The subsite affinities for the GlcNAc residues were calculated from the observed k(cat)/K-m values (A(-2), -11.0; A(-1), -1.5; A(0), -7.7; A(+1), -12.5 kJ(.)mol(-1)). The increments in the subsite affinities due to the recognition of the acetamido groups were calculated [DeltaDeltaG((N-acetyl)) = 3.3, 0, 4.0 and 0 kJ(.)mol(-1) for subsites -2, -1, 0 and +1 respectively]. The steady-state kinetic parameters for the second deacetylation reaction of (GlcNAc)(4) were also determined using (GlcNAcGlcNAcGlcNGlcNAc) as the substrate. The comparison of the experimental and theoretical values (calculated using the subsite affinities) suggests that the mono-deacetylated substrate binds strongly in a non-productive mode occupying all four subsites, thereby inhibiting the second deacetylation reaction.