Characterization of recombinant Eschericha coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase: analysis of enzymatic activity and substrate specificity

Biochem Biophys Res Commun. 1996 Nov 21;228(3):724-32. doi: 10.1006/bbrc.1996.1723.

Abstract

Recombinant E. coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (EC 3.2.2.9) was used to study the potential for this enzyme to serve as a target for chemotherapeutic intervention. An examination of the parameters required for enzymatic activity indicate that the nucleosidase functions over a broad range of pH and temperature, with acidic conditions and temperatures of 37-45 degrees C being optimal. Analogs of 5'-methylthioadenosine and adenosine were assessed as potential enzyme inhibitors and to provide details regarding substrate specificity and reaction mechanism. The 5'-arylthio analog, 5'-(p-nitrophenyl)thioadenosine, was the most potent enzyme inhibitor studied, with a Ki of 20nM. A mutant of the nucleosidase lacking the first 8 amino acids was engineered to determine the contribution of these conserved residues toward enzyme specificity. The truncated enzyme exhibited a K(m)[MTA] of 1.43 microM, approximately 3 fold higher than the K(m) reported for the full-length nucleosidase.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Escherichia coli / enzymology*
  • Kinetics
  • N-Glycosyl Hydrolases / antagonists & inhibitors
  • N-Glycosyl Hydrolases / genetics
  • N-Glycosyl Hydrolases / metabolism*
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Temperature

Substances

  • Recombinant Proteins
  • N-Glycosyl Hydrolases
  • adenosylhomocysteine nucleosidase