Alteration of aspartate 101 in the active site of Escherichia coli alkaline phosphatase enhances the catalytic activity.
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)
Chaidaroglou
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Department of Chemistry, Boston College, Chestnut Hill, MA 02167.
Kantrowitz
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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
D001224:Aspartic Acid; D000469:Alkaline Phosphatase