title: Alteration of aspartate 101 in the active site of Escherichia coli alkaline phosphatase enhances the catalytic activity.
abstract: The function of aspartic acid residue 101 in the active site of Escherichia coli alkaline phosphatase was investigated by site-specific mutagenesis. A mutant version of alkaline phosphatase was constructed with alanine in place of aspartic acid at position 101. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, pH 8.0, both the kcat and the Km for the mutant enzyme increase by approximately 2-fold, resulting in almost no change in the kcat/Km ratio. Under conditions of no external phosphate acceptor and pH 8.0, both the kcat and the Km for the mutant enzyme decrease by approximately 2-fold, again resulting in almost no change in the kcat/Km ratio. The kcat for the hydrolysis of 4-methyl-umbelliferyl phosphate and p-nitrophenyl phosphate are nearly identical for both the wild-type and mutant enzymes, as is the Ki for inorganic phosphate. The replacement of aspartic acid 101 by alanine does have a significant effect on the activity of the enzyme as a function of pH, especially in the presence of a phosphate acceptor. At pH 9.4 the mutant enzyme exhibits 3-fold higher activity than the wild-type. The mutant enzyme also exhibits a substantial decrease in thermal stability: it is half inactivated by treatment at 49 degrees C for 15 min compared to 71 degrees C for the wild-type enzyme. The data reported here suggest that this amino acid substitution alters the rates of steps after the formation of the phospho-enzyme intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, Boston College, Chestnut Hill, MA 02167.
authors.forename: A
authors.initials: A
authors.lastname: Chaidaroglou
authors.forename: E R
authors.initials: ER
authors.lastname: Kantrowitz
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics*; D001224:Aspartic Acid / Q000235:genetics*; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: D001224:Aspartic Acid; D000469:Alkaline Phosphatase