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PDBsum entry 2anh

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Alkaline phosphatase PDB id
2anh
Jmol
Contents
Protein chains
446 a.a. *
Ligands
PO4 ×3
Metals
_ZN ×6
Waters ×185
* Residue conservation analysis

References listed in PDB file
Key reference
Title Mutations at positions 153 and 328 in escherichia coli alkaline phosphatase provide insight towards the structure and function of mammalian and yeast alkaline phosphatases.
Authors J.E.Murphy, T.T.Tibbitts, E.R.Kantrowitz.
Ref. J Mol Biol, 1995, 253, 604-617. [DOI no: 10.1006/jmbi.1995.0576]
PubMed id 7473737
Abstract
In order to understand some of the differences between human placental, human, Saccharomyces cerevisiae and Escherichia coli alkaline phosphatases in specific activity, activation by magnesium, and pH versus activity profiles, the X-ray crystal structures of three mutant E. coli alkaline phosphatases have been determined. The aligned sequences of alkaline phosphatases from mammalian, yeast and E. coli show that 25 to 30% of the amino acids are absolutely conserved and the active site residues are completely conserved with the exception of residues 153, 328 and 155. The bacterial enzyme has a salt-bridge, Asp153/Lys328, near the third metal binding site which, based on sequence homology, is apparently absent in the yeast and mammalian enzymes. The human enzymes have histidine at positions 153 and 328, and the yeast enzyme has histidine at position 328. In the E. coli enzyme, Asp153 was replaced by histidine (D153H), Lys328 was replaced by histidine (K328H), and a double mutant (DM) was constructed containing both mutations. The structure of the K328H enzyme was refined using cross-validation to a resolution of 2.3 A with a working R-factor of 0.181 and a free R-factor of 0.249. The DM structure was determined to a resolution of 2.5 A with a working R-factor of 0.166 and a free R-factor of 0.233. The structure of the D135H enzyme, which has been reported to a resolution of 2.4 A, has been re-refined using cross-validation to a working R-factor of 0.179 and a free R-factor of 0.239 for controlled comparisons with the two new structures. In all three structures the most significant changes are related to the bound phosphate inhibitor and the identity of the metal ion in the third binding site. The changes in the position of the phosphate group and the alterations at the third metal binding site indicate the structural basis for the variations in the steady-state kinetic parameters previously reported for these enzymes.
Figure 2.
Figure 2. The active site region of E. coli alkaline phosphatase including the phosphate group, magnesium ion and two zinc-binding sites. Not all the ligands are shown. Water molecules are indicated by the letter w. Hydrogen bonds are shown as broken lines (Kim & Wyckoff, 1991).
Figure 9.
Figure 9. Stereoview comparing the wild-type (thinnest lines), the K328H (middle lines) and the DM (thickest lines) structures. This view focuses on the new anion binding site (PO4-B) around Tyr169. This site exists in the D153H and DM structures, but is not seen in the K328H or the wild-type structure. The Figure shows the loop region formed by the disulfide bridge between Cys168 and Cys178. The conformation of this loop region appears to be conserved in all three structures.
The above figures are reprinted by permission from Elsevier: J Mol Biol (1995, 253, 604-617) copyright 1995.
Secondary reference #1
Title Conversion of a magnesium binding site into a zinc binding site by a single amino acid substitution in escherichia coli alkaline phosphatase.
Authors J.E.Murphy, X.Xu, E.R.Kantrowitz.
Ref. J Biol Chem, 1993, 268, 21497-21500.
PubMed id 8407998
Abstract
Secondary reference #2
Title Reaction mechanism of alkaline phosphatase based on crystal structures. Two-Metal ion catalysis.
Authors E.E.Kim, H.W.Wyckoff.
Ref. J Mol Biol, 1991, 218, 449-464.
PubMed id 2010919
Abstract
PROCHECK
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