Characterization of recombinant Eschericha coli 5'-Methylthioadenosine/S-Adenosylhomocysteine nucleosidase: Analysis of enzymatic activity and substrate specificity

Kenneth A. Cornell, William E. Swarts, Ronald D. Barry, Michael K. Riscoe

Research output: Contribution to journalArticle

64 Scopus citations

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°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 K(i) of 20nM. A mutant of the nucleosidase lacking the first 8 amino acids was engineered to determine tile contribution of these conserved residues toward enzyme specificity. The truncated enzyme exhibited a K(m[MTA]) of 1.43 μM, approximately 3 fold higher than the K(m) reported for the full-length nucleosidase.

Original languageEnglish (US)
Pages (from-to)724-732
Number of pages9
JournalBiochemical and Biophysical Research Communications
Volume228
Issue number3
DOIs
StatePublished - Nov 21 1996

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ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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