Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis.
Alkaline phosphatase (AP) is a widely distributed non-specific phosphomonoesterase that functions through formation of a covalent phosphoseryl intermediate (E-P). The enzyme also catalyzes phosphoryl transfer reaction to various alcohols. Escherichia coli AP is a homodimer with 449 residues per monomer. It is a metalloenzyme with two Zn2+ and one Mg2+ at each active site. The crystal structure of native E. coli AP complexed with inorganic phosphate (Pi), which is a strong competitive inhibitor as well as a substrate for the reverse reaction, has been refined at 2.0 A resolution. Some parts of the molecular have been retraced, starting from the previous 2.8 A study. The active site has been modified substantially and is described in this paper. The changes in the active site region suggest the need to reinterpret earlier spectral data, and suggestions are made. Also presented are the structures of the Cd-substituted enzyme complexed with inorganic phosphate at 2.5 A resolution, and the phosphate-free native enzyme at 2.8 A resolution. At pH 7.5, where the X-ray data were collected, the Cd-substituted enzyme is predominantly the covalent phosphoenzyme (E-P) while the native Zn/Mg enzyme exists in predominantly noncovalent (E.P) form. Implication of these results for the catalytic mechanism of the enzyme is discussed. APs from other sources are believed to function in a similar manner.
Kim
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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Wyckoff
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HW
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D006860:Hydrogen Bonding; D008274:Magnesium / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D014961:X-Ray Diffraction; D015032:Zinc / Q000378:metabolism*
D010710:Phosphates; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc