title: Effect of a T81A mutation at the subunit interface on catalytic properties of alkaline phosphatase from Escherichia coli.
abstract: Although alkaline phosphatase (APase) from Escherichia coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics supported by a model describing a dimeric enzyme with conformationally and kinetically non-equivalent subunits. The proposed model, explaining the mechanism of substrate hydrolysis, encompasses a conformational change mediated by subunit interactions [S. Orhanović, M. Pavela-Vrancic, Eur. J. Biochem. 270 (2003) 4356-4364]. The significance of interactions at the subunit interface and the involvement of the beta-pleated sheet stretching from underneath the active site to the subunit surface, in the catalytic mechanism, has been probed by site-directed mutagenesis. The mutant APase, carrying alanine in place of Thr81, was analyzed in comparison to the wild-type protein. The T81A mutation, introduced at the subunit interface, significantly affected the protein kinetic properties, emphasizing the importance of subunit interactions in the catalytic process.
authors.affiliation: Department of Chemistry, Faculty of Natural Sciences, Mathematics and Education, University of Split, N. Tesle 12, 21000 Split, Croatia. stipe@pmfst.hr
authors.forename: S
authors.initials: S
authors.lastname: Orhanović
authors.forename: V
authors.initials: V
authors.lastname: Bucević-Popović
authors.forename: M
authors.initials: M
authors.lastname: Pavela-Vrancic
authors.forename: D
authors.initials: D
authors.lastname: Vujaklija
authors.forename: V
authors.initials: V
authors.lastname: Gamulin
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019943:Amino Acid Substitution; D020134:Catalytic Domain / Q000235:genetics; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase