spacer
spacer

PDBsum entry 1ed8

Go to PDB code: 
protein ligands metals Protein-protein interface(s) links
Hydrolase PDB id
1ed8
Jmol
Contents
Protein chains
449 a.a. *
Ligands
PO4 ×2
SO4 ×2
Metals
_ZN ×6
_MG ×2
Waters ×604
* Residue conservation analysis
PDB id:
1ed8
Name: Hydrolase
Title: Structure of e. Coli alkaline phosphatase inhibited by the inorganic phosphate at 1.75a resolution
Structure: Alkaline phosphatase. Chain: a, b. Engineered: yes
Source: Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PQS)
Resolution:
1.75Å     R-factor:   0.197     R-free:   0.228
Authors: B.Stec,K.M.Holtz,E.R.Kantrowitz
Key ref:
B.Stec et al. (2000). A revised mechanism for the alkaline phosphatase reaction involving three metal ions. J Mol Biol, 299, 1303-1311. PubMed id: 10873454 DOI: 10.1006/jmbi.2000.3799
Date:
27-Jan-00     Release date:   20-Sep-00    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P00634  (PPB_ECOLI) -  Alkaline phosphatase
Seq:
Struc:
471 a.a.
449 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.3.1.3.1  - Alkaline phosphatase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: A phosphate monoester + H2O = an alcohol + phosphate
phosphate monoester
+ H(2)O
= alcohol
+
phosphate
Bound ligand (Het Group name = PO4)
corresponds exactly
      Cofactor: Magnesium; Zinc
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     periplasmic space   2 terms 
  Biological process     metabolic process   3 terms 
  Biochemical function     catalytic activity     10 terms  

 

 
    reference    
 
 
DOI no: 10.1006/jmbi.2000.3799 J Mol Biol 299:1303-1311 (2000)
PubMed id: 10873454  
 
 
A revised mechanism for the alkaline phosphatase reaction involving three metal ions.
B.Stec, K.M.Holtz, E.R.Kantrowitz.
 
  ABSTRACT  
 
Here, X-ray crystallography has been used to investigate the proposed double in-line displacement mechanism of Escherichia coli alkaline phosphatase in which two of the three active-site metal ions have a direct role in catalysis. Two new X-ray crystal structures of the wild-type enzyme in the absence and presence of inorganic phosphate have been refined at 1.75 A to final working R-factors of 15.4% and 16.4%, respectively. In the refinement of both structures, residues in the active sites were treated anisotropically. The ellipsoids resulting from the partial anisotropic refinement show a clear route for the binding and release of substrate/product. In addition, a direct comparison of the refined structures with and without phosphate reveal a strong correlation between the occupancy of the third metal-binding site and the conformation of the Ser102 nucleophile. These findings clarify two important and unresolved aspects of the previously proposed catalytic mechanism, how Ser102 is activated for nucleophilic attack and why a magnesium ion in the third metal site is required for catalysis. Analysis of these results suggest that three metal-ion assisted catalysis is a more accurate description of the mechanism of the alkaline phosphatase reaction. A revised mechanism for the catalytic reaction of alkaline phosphatase is proposed on the basis of the two new X-ray crystal structures reported.
 
  Selected figure(s)  
 
Figure 1.
Figure 1. Stereoview of the 2F[o] - F[c] electron density maps (s = 1.5) with the representative atomic model at the active sites of the (a) AP_Pi and (b) AP_noPi structures. The A subunit is shown.
Figure 3.
Figure 3. The behavior of the R[4]s (continuous line), R[FREE] (broken line) and R[ALL] (dash-dot-dot) at different stages of the refinement with SHELXL-97 (Sheldrick & Schneider, 1997) for the (a) AP_Pi and (b) AP_noPi structures. The five refinement stages are: (1) protein alone; (2) protein, metals and phosphate; (3) protein, metals, phosphate and 50 % of the water molecules; (4) protein, metals, phosphate and all the water molecules; and (5) protein, metals, phosphate, waters plus an isotropic refinement of the active-site atoms.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 299, 1303-1311) copyright 2000.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21512229 S.Sekiguchi, H.Kohno, K.Yasukawa, and K.Inouye (2011).
Chemiluminescent enzyme immunoassay for measuring leptin.
  Biosci Biotechnol Biochem, 75, 752-756.  
20854710 W.Yang (2011).
Nucleases: diversity of structure, function and mechanism.
  Q Rev Biophys, 44, 1.  
20300652 D.E.Almonacid, E.R.Yera, J.B.Mitchell, and P.C.Babbitt (2010).
Quantitative comparison of catalytic mechanisms and overall reactions in convergently evolved enzymes: implications for classification of enzyme function.
  PLoS Comput Biol, 6, e1000700.  
19916164 D.Koutsioulis, A.Lyskowski, S.Mäki, E.Guthrie, G.Feller, V.Bouriotis, and P.Heikinheimo (2010).
Coordination sphere of the third metal site is essential to the activity and metal selectivity of alkaline phosphatases.
  Protein Sci, 19, 75-84.
PDB codes: 2w5v 2w5w 2w5x
20135039 D.R.Edwards, W.Y.Tsang, A.A.Neverov, and R.S.Brown (2010).
On the question of stepwise vs. concerted cleavage of RNA models promoted by a synthetic dinuclear Zn(II) complex in methanol: implementation of a noncleavable phosphonate probe.
  Org Biomol Chem, 8, 822-827.  
20057143 H.Tsuruta, B.Mikami, T.Higashi, and Y.Aizono (2010).
Crystal structure of cold-active alkaline phosphatase from the psychrophile Shewanella sp.
  Biosci Biotechnol Biochem, 74, 69-74.
PDB code: 3a52
19368558 P.O.Heidarsson, S.T.Sigurdsson, and B.Asgeirsson (2009).
Structural features and dynamics of a cold-adapted alkaline phosphatase studied by EPR spectroscopy.
  FEBS J, 276, 2725-2735.  
18604568 C.Andreini, I.Bertini, G.Cavallaro, G.L.Holliday, and J.M.Thornton (2008).
Metal ions in biological catalysis: from enzyme databases to general principles.
  J Biol Inorg Chem, 13, 1205-1218.  
18851975 J.G.Zalatan, T.D.Fenn, and D.Herschlag (2008).
Comparative enzymology in the alkaline phosphatase superfamily to determine the catalytic role of an active-site metal ion.
  J Mol Biol, 384, 1174-1189.
PDB code: 3dyc
18294135 L.P.Xie, G.R.Xu, W.Z.Cao, J.Zhang, and R.Q.Zhang (2008).
An essential tryptophan residue in alkaline phosphatase from pearl oyster (Pinctada fucata).
  Biochemistry (Mosc), 73, 87-91.  
18627128 P.J.O'Brien, J.K.Lassila, T.D.Fenn, J.G.Zalatan, and D.Herschlag (2008).
Arginine coordination in enzymatic phosphoryl transfer: evaluation of the effect of Arg166 mutations in Escherichia coli alkaline phosphatase.
  Biochemistry, 47, 7663-7672.
PDB code: 3cmr
18833291 S.Gong, and T.L.Blundell (2008).
Discarding functional residues from the substitution table improves predictions of active sites within three-dimensional structures.
  PLoS Comput Biol, 4, e1000179.  
18365148 Y.Zhang, C.Ji, X.Zhang, Z.Yang, J.Peng, R.Qiu, Y.Xie, and Y.Mao (2008).
A moderately thermostable alkaline phosphatase from Geobacillus thermodenitrificans T2: cloning, expression and biochemical characterization.
  Appl Biochem Biotechnol, 151, 81-92.  
17893790 R.Mitra, M.W.Peters, and M.J.Scott (2007).
Synthesis and reactivity of a C3-symmetric trinuclear zinc(II) hydroxide catalyst efficient at phosphate diester transesterification.
  Dalton Trans, (), 3924-3935.  
  18404473 J.L.Millán (2006).
Alkaline Phosphatases : Structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes.
  Purinergic Signal, 2, 335-341.  
17008720 J.Wang, and E.R.Kantrowitz (2006).
Trapping the tetrahedral intermediate in the alkaline phosphatase reaction by substitution of the active site serine with threonine.
  Protein Sci, 15, 2395-2401.
PDB codes: 2g9y 2ga3
16815919 P.Llinas, M.Masella, T.Stigbrand, A.Ménez, E.A.Stura, and M.H.Le Du (2006).
Structural studies of human alkaline phosphatase in complex with strontium: implication for its secondary effect in bones.
  Protein Sci, 15, 1691-1700.
PDB code: 2glq
17109483 X.Huang, X.E.Zhang, Y.F.Zhou, Z.P.Zhang, and A.E.Cass (2006).
Directed evolution of the 5'-untranslated region of the phoA gene in Escherichia coli simultaneously yields a stronger promoter and a stronger Shine-Dalgarno sequence.
  Biotechnol J, 1, 1275-1282.  
15833276 H.T.Chen, L.P.Xie, Z.Y.Yu, G.R.Xu, and R.Q.Zhang (2005).
Chemical modification studies on alkaline phosphatase from pearl oyster (Pinctada fucata): a substrate reaction course analysis and involvement of essential arginine and lysine residues at the active site.
  Int J Biochem Cell Biol, 37, 1446-1457.  
16092891 J.L.Hougland, A.V.Kravchuk, D.Herschlag, and J.A.Piccirilli (2005).
Functional identification of catalytic metal ion binding sites within RNA.
  PLoS Biol, 3, e277.  
15973058 M.Ishibashi, S.Yamashita, and M.Tokunaga (2005).
Characterization of halophilic alkaline phosphatase from Halomonas sp. 593, a moderately halophilic bacterium.
  Biosci Biotechnol Biochem, 69, 1213-1216.  
15885097 T.Harada, I.Koyama, T.Matsunaga, A.Kikuno, T.Kasahara, M.Hassimoto, D.H.Alpers, and T.Komoda (2005).
Characterization of structural and catalytic differences in rat intestinal alkaline phosphatase isozymes.
  FEBS J, 272, 2477-2486.  
16328740 Y.Zhu, X.Y.Song, W.H.Zhao, and Y.X.Zhang (2005).
Effects of magnesium ions on thermal inactivation of alkaline phosphatase.
  Protein J, 24, 479-485.  
15477392 M.Brännvall, E.Kikovska, and L.A.Kirsebom (2004).
Cross talk between the +73/294 interaction and the cleavage site in RNase P RNA mediated cleavage.
  Nucleic Acids Res, 32, 5418-5429.  
15333925 M.M.de Backer, S.McSweeney, P.F.Lindley, and E.Hough (2004).
Ligand-binding and metal-exchange crystallographic studies on shrimp alkaline phosphatase.
  Acta Crystallogr D Biol Crystallogr, 60, 1555-1561.
PDB codes: 1shn 1shq
14978036 S.W.Nelson, R.B.Honzatko, and H.J.Fromm (2004).
Origin of cooperativity in the activation of fructose-1,6-bisphosphatase by Mg2+.
  J Biol Chem, 279, 18481-18487.  
14557260 C.Zambonelli, M.Casali, and M.F.Roberts (2003).
Mutagenesis of putative catalytic and regulatory residues of Streptomyces chromofuscus phospholipase D differentially modifies phosphatase and phosphodiesterase activities.
  J Biol Chem, 278, 52282-52289.  
12595528 J.Y.Choe, C.V.Iancu, H.J.Fromm, and R.B.Honzatko (2003).
Metaphosphate in the active site of fructose-1,6-bisphosphatase.
  J Biol Chem, 278, 16015-16020.
PDB codes: 1nuw 1nux 1nuy
12707276 R.R.Boulanger, and E.R.Kantrowitz (2003).
Characterization of a monomeric Escherichia coli alkaline phosphatase formed upon a single amino acid substitution.
  J Biol Chem, 278, 23497-23501.  
14622301 S.Orhanović, and M.Pavela-Vrancic (2003).
Dimer asymmetry and the catalytic cycle of alkaline phosphatase from Escherichia coli.
  Eur J Biochem, 270, 4356-4364.  
12399456 C.L.Wojciechowski, and E.R.Kantrowitz (2002).
Altering of the metal specificity of Escherichia coli alkaline phosphatase.
  J Biol Chem, 277, 50476-50481.  
11910033 C.L.Wojciechowski, J.P.Cardia, and E.R.Kantrowitz (2002).
Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity.
  Protein Sci, 11, 903-911.  
11863460 P.J.O'Brien, and D.Herschlag (2002).
Alkaline phosphatase revisited: hydrolysis of alkyl phosphates.
  Biochemistry, 41, 3207-3225.  
12036047 T.Murakawa, H.Yamagata, H.Tsuruta, and Y.Aizono (2002).
Cloning of cold-active alkaline phosphatase gene of a psychrophile, Shewanella sp., and expression of the recombinant enzyme.
  Biosci Biotechnol Biochem, 66, 754-761.  
12127574 Y.X.Zhang, Y.Zhu, H.W.Xi, Y.L.Liu, and H.M.Zhou (2002).
Refolding and reactivation of calf intestinal alkaline phosphatase with excess magnesium ions.
  Int J Biochem Cell Biol, 34, 1241-1247.  
11828484 B.H.Muller, C.Lamoure, M.H.Le Du, L.Cattolico, E.Lajeunesse, F.Lemaître, A.Pearson, F.Ducancel, A.Ménez, and J.C.Boulain (2001).
Improving Escherichia coli alkaline phosphatase efficacy by additional mutations inside and outside the catalytic pocket.
  Chembiochem, 2, 517-523.  
11266592 H.C.Hung, and G.G.Chang (2001).
Differentiation of the slow-binding mechanism for magnesium ion activation and zinc ion inhibition of human placental alkaline phosphatase.
  Protein Sci, 10, 34-45.  
11721007 H.C.Hung, and G.G.Chang (2001).
Multiple unfolding intermediates of human placental alkaline phosphatase in equilibrium urea denaturation.
  Biophys J, 81, 3456-3471.  
11170378 K.A.Johnson, L.Chen, H.Yang, M.F.Roberts, and B.Stec (2001).
Crystal structure and catalytic mechanism of the MJ0109 gene product: a bifunctional enzyme with inositol monophosphatase and fructose 1,6-bisphosphatase activities.
  Biochemistry, 40, 618-630.
PDB codes: 1g0h 1g0i
11800017 Y.D.Park, Y.Yang, Q.X.Chen, H.N.Lin, Q.Liu, and H.M.Zhou (2001).
Kinetics of complexing activation by the magnesium ion on green crab (Scylla serrata) alkaline phosphatase.
  Biochem Cell Biol, 79, 765-772.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.