title: Studies on the interaction of Escherichia coli agmatinase with manganese ions: structural and kinetic studies of the H126N and H151N variants.
abstract: The H126N and H151N variants of Escherichia coli agmatinase (EC 3.5.3.11) were produced by site-directed mutagenesis, and their kinetic and structural properties were examined. About 51% and 30% of wild-type activity were expressed by fully manganese activated species of the H126N and H151N variants, respectively. Mutations were not accompanied by changes in the K(m) value for arginine (1.2+/-0.3 mM), K(i) value for putrescine inhibition (3.2+/-0.4 mM), molecular weight (M(r) 67,000+/-2000), tryptophan fluorescence properties (lambda(max) = 342 nm) or CD spectra of the enzyme. However, the interaction with the required manganese ions was significantly altered, as indicated by the effects of dialysis of the enzymes against metal-free buffer. We conclude that replacement of His151 with asparagine results in the loss of a catalytically essential Mn(2+) upon dialysis and concomitant reversible inactivation of the H151N mutant, and that the affinity of a more weakly bound Mn(2+) is decreased in the H126N variant.
authors.affiliation: Departamento de Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
authors.forename: Mónica
authors.initials: M
authors.lastname: Salas
authors.forename: Vasthi
authors.initials: V
authors.lastname: López
authors.forename: Elena
authors.initials: E
authors.lastname: Uribe
authors.forename: Nelson
authors.initials: N
authors.lastname: Carvajal
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008345:Manganese / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics; D014512:Ureohydrolases / Q000737:chemistry* / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D008345:Manganese; D014512:Ureohydrolases; C019625:agmatinase