title: Enhanced catalysis by active-site mutagenesis at aspartic acid 153 in Escherichia coli alkaline phosphatase.
abstract: Bacterial alkaline phosphatase catalyzes the hydrolysis and transphosphorylation of phosphate monoesters. Site-directed mutagenesis was used to change the active-site residue Asp-153 to Ala and Asn. In the wild-type enzyme Asp-153 forms a second-sphere complex with Mg2+. The activity of mutant enzymes D153N and D153A is dependent on the inclusion of Mg2+ in the assay buffer. The steady-state kinetic parameters of the D153N mutant display small enhancements, relative to wild type, in buffers containing 10 mM Mg2+. In contrast, the D153A mutation gives rise to a 6.3-fold increase in kcat, a 13.7-fold increase in kcat/Km (50 mM Tris, pH 8), and a 159-fold increase in Ki for Pi (1 M Tris, pH 8). In addition, the activity of D153A increases 25-fold as the pH is increased from 7 to 9. D153A hydrolyzes substrates with widely differing pKa's of their phenolic leaving groups (PNPP and DNPP), at similar rates. As with wild type, the rate-determining step takes place after the initial nucleophilic displacement (k2). The increase in kcat for the D153A mutant indicates that the rate of release of phosphate from the enzyme product complex (k4) has been enhanced.
authors.affiliation: Department of Chemistry, Oberlin College, Ohio 44074.
authors.forename: A R
authors.initials: AR
authors.lastname: Matlin
authors.forename: D A
authors.initials: DA
authors.lastname: Kendall
authors.forename: K S
authors.initials: KS
authors.lastname: Carano
authors.forename: J A
authors.initials: JA
authors.lastname: Banzon
authors.forename: S B
authors.initials: SB
authors.lastname: Klecka
authors.forename: N M
authors.initials: NM
authors.lastname: Solomon
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D001224:Aspartic Acid; D000469:Alkaline Phosphatase; D008274:Magnesium