Probing the role of histidine-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase by site-specific mutagenesis.
In the X-ray structure of Escherichia coli alkaline phosphatase at 2.0-A resolution, His-372 was found only 3.8 A away from the zinc and forms a hydrogen-bonding interaction with Asp-327, a bidentate ligand of the zinc at the M1 site. However, His-372 does not directly interact with the zinc atom at the M1 site. In order to investigate the role of the side chain of His-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase, site-directed mutagenesis was used to convert His-372 to alanine. The fact that the His-372-->Ala enzyme has similar zinc binding affinity as the wild-type enzyme indicates that His-372 is not involved in zinc binding at the M1 site. However, the altered kinetic behavior of the mutant enzyme compared to the wild-type enzyme suggests that the imidazole ring of His-372 plays an indirect role in the catalytic mechanism of the enzyme. The hydrolysis activity of the His-372-->Ala enzyme at pH 8.0 is 10-fold lower than that of the wild-type enzyme. In the presence of a phosphate acceptor at pH 8.0, the mutant enzyme is approximately 80% as active as the wild-type enzyme. Therefore, the His-372-->Ala mutation selectively enhances the transphosphorylation activity of the enzyme. The His-372-->Ala enzyme also exhibits 4- and 30-fold decreases in Km as compared to the wild-type enzyme in 0.1 M MOPS buffer and 1.0 M Tris, buffer at pH 8.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Chemistry, Boston College, Massachusetts 02167.
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D000409:Alanine / Q000737:chemistry; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D013379:Substrate Specificity; D014961:X-Ray Diffraction; D015032:Zinc / Q000378:metabolism*
D010710:Phosphates; D006639:Histidine; D000469:Alkaline Phosphatase; D015032:Zinc; D000409:Alanine