Roles of the exposed aromatic residues in crystalline chitin hydrolysis bychitinase a from Serratia marcescens 2170

Four exposed aromatic residues, two in the N-terminal domain (Trp-69 and Tr p-33) and two in the catalytic domain (Trp-245 and Phe-232) of Serratia mar cescens chitinase A, are linearly aligned with the deep catalytic cleft. To investigate the importance of these residues in the binding activity and h ydrolyzing activity against insoluble chitin, site-directed mutagenesis to alanine was carried out. The substitution of Trp-69, Trp-33, or Trp-245 sig nificantly reduced the binding activity to both highly crystalline beta -ch itin and colloidal chitin. The substitution of Phe-232, which is located cl osest to the catalytic cleft, did not affect the binding activity. On the o ther hand, the hydrolyzing activity against beta -chitin microfibrils was s ignificantly reduced by the substitution of any one of the four aromatic re sidues including Phe-232. None of the mutations reduced the hydrolyzing act ivity against soluble substrates. These results clearly demonstrate that th e four exposed aromatic residues are essential determinants for crystalline chitin hydrolysis. Three of them, two in the N-terminal domain and one in the catalytic domain, play vital roles in the chitin binding. Phe-232 appea red to be important for guiding the chitin chain into the catalytic cleft. Based on these observations, a model for processive hydrolysis of crystalli ne chitin by chitinase A is proposed.