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

Biochem J. 2003 Nov 15;376(Pt 1):237-44. doi: 10.1042/BJ20030419.


Bacillus circulans chitinase A1 (ChiA1) 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 beta-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 ChiA1 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 kcat-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids, Aromatic / genetics
  • Amino Acids, Aromatic / physiology*
  • Bacillus / enzymology*
  • Binding Sites
  • Catalytic Domain
  • Chitin / chemistry
  • Chitin / metabolism*
  • Chitinases / chemistry*
  • Chitinases / genetics
  • Chitinases / metabolism*
  • Crystallization
  • Hydrolysis
  • Mutagenesis, Site-Directed
  • Tryptophan / genetics
  • Tyrosine / genetics


  • Amino Acids, Aromatic
  • Chitin
  • Tyrosine
  • Tryptophan
  • Chitinases