Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate-assisted catalysis by orienting the 2-acetamido group and stabilizing the transition state.

摘要: SpHex, a retaining family 20 glycosidase from Streptomyces plicatus, catalyzes the hydrolysis of N-acetyl-β-hexosaminides. Accumulating evidence suggests that hydrolytic mechanism involves substrate-assisted catalysis wherein 2-acetamido substituent acts as nucleophile to form an oxazolinium ion intermediate. The role conserved aspartate residue (D313) in active site ofSpHex was investigated through kinetic and structural analyses two variant enzymes, D313A D313N. Three-dimensional structures wild-type enzymes product complexes with N-acetyl-d-glucosamine revealed substantial differences. In group found conformations which only one is able aid anchimeric assistance. mutation D313N results steric clash between Asn-313 preventing providing assistance, consistent large reduction catalytic efficiency insensitivity this chemical rescue. By comparison, shift pH optimum modest decrease activity can be rescued by using azide exogenous nucleophile. These data provide Asp-313 stabilizes transition states flanking oxazoline intermediate also assists correctly orient for catalysis. Based on analogous residues 18 chitinases 56 hyaluronidases, roles played likely general all hexosaminidases involving