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

J Biol Chem. 2002 Oct 18;277(42):40055-65. doi: 10.1074/jbc.M206481200. Epub 2002 Aug 8.


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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Aspartic Acid / chemistry*
  • Aspartic Acid / metabolism
  • Azides / chemistry
  • Catalysis
  • Dose-Response Relationship, Drug
  • Electrons
  • Hyaluronoglucosaminidase / metabolism
  • Hydrogen-Ion Concentration
  • Ions
  • Kinetics
  • Models, Chemical
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oxazoles / chemistry
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid
  • Streptomyces / enzymology*
  • beta-N-Acetylhexosaminidases / chemistry*


  • Azides
  • Ions
  • Oxazoles
  • Aspartic Acid
  • Hyaluronoglucosaminidase
  • beta-N-Acetylhexosaminidases

Associated data

  • PDB/1M01
  • PDB/1M03
  • PDB/1M04