Structures of chitobiase mutants complexed with the substrate di-N-acetyl-D-glucosamine: The catalytic role of the conserved acidic pair, aspartate 539 and glutamate 540

The catalytic domain of chitobiase (beta-N-1-4 acetylhexosaminidase) from S erratia marcescens, is an alpha/beta TIM-barrel. This enzyme belongs to fam ily 20 of glycosyl hydrolases in which a conserved amino acid pair, asparta te-glutamate, is present (Asp539-Glu540). It was proposed that catalysis by this enzyme family is carried out by glutamate 540 acting as a proton dono r and by the acetamido group of the substrate as a nucleophile. We investig ated the role of Asp539 and Glu540 by site-directed mutagenesis, biochemica l characterization and by structural analyses of chitobiase substrate co-cr ystals. We found that both residues are essential for chitoblase activity. The mutations, however, led to subtle changes in the catalytic site. Our re sults support the model that Glu540 acts as the proton donor and that Asp53 9 acts in several different ways. Asp539 restrains the acetamido group of t he substrate in a specific orientation by forming a hydrogen bond with N2 o f the non-reduced (-1) sugar. In addition, this residue participates in sub strate binding. It is also required for the correct positioning of Glu540 a nd may provide additional negative charge at the active site. Thus, these b iochemical and structural studies provide a molecular explanation for the f unctional importance and conservation of these residues. (C) 2000 Academic Press.