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

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'-(b-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 the contribution of these conserved residues reward enzyme specificity. The truncated enzyme exhibited a K-m[MTA] of 1.43 mu M, approximately 3 fold higher than the K-m reported for the full-length nucleosidase. (C) 1996 Academic Press, Inc.