title: Characterization of recombinant Eschericha coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase: analysis of enzymatic activity and substrate specificity.
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.
authors.affiliation: Department of Biochemistry, Oregon Health Sciences University, Portland 97201, USA.
authors.forename: K A
authors.initials: KA
authors.lastname: Cornell
authors.forename: W E
authors.initials: WE
authors.lastname: Swarts
authors.forename: R D
authors.initials: RD
authors.lastname: Barry
authors.forename: M K
authors.initials: MK
authors.lastname: Riscoe
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D009699:N-Glycosyl Hydrolases; C019606:adenosylhomocysteine nucleosidase