Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin

Bacillus circulans chitinase Al (ChiAl) has a deep substrate-binding cleft on top of its (beta/alpha)(8)-barrel catalytic domain and an interaction between the aromatic residues in this cleft and bound oligosaccharide has been suggested. To study the roles of these aromatic residues, especially in crystalline-chitin hydrolysis, site-directed mutagenesis of these residues was carried out. Y56A and W53A mutations at subsites -5 and -3, respectively, selectively decreased the hydrolysing activity against highly crystalline beta-chitin. W164A and W285A mutations at subsites + 1 and + 2, respectively, decreased the hydrolysing activity against crystalline P-chitin and colloidal chitin, but enhanced the activities against soluble substrates. These mutations increased the K-m-value when reduced (GlcNAc)(5) (where GlcNAc is N-acetylglucosamine) was used as the substrate, but decreased substrate inhibition observed with wild-type ChiAl at higher concentrations of this substrate. In contrast with the selective effect of the other mutations, mutations of W433 and Y279 at subsite -1 decreased the hydrolysing activity drastically against all substrates and reduced the k(cat)-value, measured with 4-methylumbelliferyl chitotrioside to 0.022% and 0.59% respectively. From these observations, it was concluded that residues Y56 and W53 are only essential for crystalline-chitin hydrolysis. W164 and W285 are very important for crystalline-chitin hydrolysis and also participate in hydrolysis of other substrates. W433 and Y279 are both essential for catalytic reaction as predicted from the structure.