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PDBsum entry 1alj

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Hydrolase (phosphoric monoester) PDB id
1alj
Jmol
Contents
Protein chains
446 a.a. *
Ligands
PO4 ×2
Metals
_ZN ×2
_MG ×2
Waters ×219
* Residue conservation analysis

References listed in PDB file
Key reference
Title Escherichia coli alkaline phosphatase: X-Ray structural studies of a mutant enzyme (his-412-->Asn) at one of the catalytically important zinc binding sites.
Authors L.Ma, T.T.Tibbitts, E.R.Kantrowitz.
Ref. Protein Sci, 1995, 4, 1498-1506. [DOI no: 10.1002/pro.5560040807]
PubMed id 8520475
Abstract
The X-ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His-412 was replaced by Asn has been determined at both low (-Zn) and high (+Zn) concentrations of zinc. In the wild-type structure, His-412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn1) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614-31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild-type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N (+Zn) enzyme was refined to an R-factor of 0.174 at 2.2 A resolution, and the structure of the H412N(-Zn) enzyme was refined to an R-factor of 0.166 at a resolution of 2.6 A. Both indicated that the Asn residue substituted for His-412 did not coordinate well to Zn1. In the H412N(-Zn) structure, the Zn1 site had very low occupancy and the phosphate was shifted by 1.8 A from its position in the wild-type structure. The Mg binding site was also affected by the substitution of Asn for His-412. Both structures of the H412N enzyme also revealed a surface-accessible cavity near the Zn1 site that may serve as a binding site for Tris.(ABSTRACT TRUNCATED AT 250 WORDS)
Figure 1.
ig. 1. Active site of E. coli alkalinephosphatase. Shown are n,, Zn2, phosphate (Pi),and side-chain ligands. The side chain of Asp-51, which bridges the Mg nd Zn2 sites, is lso shown here; for clarity, the other ligands o Mg are not shown. Zn, interacts with an im- idazole nitrogen of is-412 and His-33, one of he phosphate oxygens, and thearboxylate oxygens of Asp-327. Zn,is oordinated to Asp- His-370, hosphate, and Also shown s Ser-102, which s during the reaction, and which interacts with thephosphate.
Figure 4.
Fig. 4. Ramachandranplot of dihedralanglesfortheH412N(+Zn) structure.Datashowthat 90.6% ofthe892residuesare in themostfa- voredregions (A, B, L), 9.170 intheaditionalallowedregions(a,b, I, p),andonly 0.3% in thedisallowedregions.Thisfigure was prepared usingPROCHECK(Laskowskiet al.. 1993).
The above figures are reprinted from an Open Access publication published by the Protein Society: Protein Sci (1995, 4, 1498-1506) copyright 1995.
Secondary reference #1
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
Secondary reference #2
Title Mutations at histidine 412 alter zinc binding and eliminate transferase activity in escherichia coli alkaline phosphatase.
Authors L.Ma, E.R.Kantrowitz.
Ref. J Biol Chem, 1994, 269, 31614-31619.
PubMed id 7989332
Abstract
PROCHECK
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 Headers