A water-mediated salt link in the catalytic site of Escherichia coli alkaline phosphatase may influence activity.
Escherichia coli alkaline phosphatase catalyzes the hydrolysis of a wide variety of phosphomonoesters at similar rates, and the reaction proceeds through a phosphoenzyme intermediate. The active site region is highly conserved between the E. coli and mammalian alkaline phosphatases. The three-dimensional structure of the E. coli enzyme indicates that Lys-328, which is replaced by histidine in all mammalian alkaline phosphatases, is bridged to the phosphate through a water molecule. This water molecule is also hydrogen bonded to Asp-327, a bidendate ligand of the one of the two zinc atoms. Here we report the use of site-specific mutagenesis to convert Lys-328 to both histidine and alanine. Steady-state kinetic studies above pH 7.0 indicate that both mutant enzymes have altered pH versus activity profiles compared to the profile for the wild-type enzyme. At pH 10.3, in the presence of Tris, the Lys-328----Ala enzyme is approximately 14-fold more active than the wild-type enzyme. At the same pH in the absence of Tris the Lys-328----Ala enzyme is still 6-fold more active than the wild-type enzyme. Both mutant enzymes have lower phosphate affinities than the wild-type enzyme at all pH values investigated. Pre-steady-state kinetics at pH 5.5 reveal that the Lys-328----Ala enzyme behaves very similar to the phosphate-free wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02167.
Kantrowitz
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D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012492:Salts; D014867:Water; D015032:Zinc / Q000378:metabolism
D012492:Salts; D014867:Water; D000469:Alkaline Phosphatase; D015032:Zinc; D008239:Lysine