The essential acidic amino acid residues for catalytic activity of an exo-β- d-glucosaminidase from Trichoderma reesei

Abstract

In order to investigate the residues important for the catalysis of an exo-β- d-glucosaminidase from Trichoderma reesei (Gls93), we performed site-directed mutagenesis study on this enzyme. Seven acidic amino acid residues in putative catalytic TIM domain, Glu409, Glu415, Asp464, Glu470, Asp513, Glu539 and Asp655, were selected for the mutational studies, each of which is located close to a C-terminus of the predicted β-strands and conserved within exo-β- d-glucosaminidase (GlcNase) subgroup of glycoside hydrolase family (GHF) 2 except for Glu470. The mutants of Gls93 at the residue Asp464 and/or Glu539, which showed a similar CD spectrum pattern to that of the wild-type enzyme, hardly have activity, indicating that Asp464 and Glu539 function as the catalytic residues of Gls93. Furthermore, the sterically conserved mutation of Glu409, Glu415 and Asp513 (the mutants E409Q, E415Q and D513N, respectively) gave rise to the change in its kinetic parameters, suggesting that these carboxylic residues could contribute to substrate recognition/binding or arrangement of ionic environment of the catalytic residues.