PDBsum entry 1urb

Go to PDB code: 
protein ligands metals Protein-protein interface(s) links
Alkaline phosphatase PDB id
Protein chains
446 a.a. *
PO4 ×4
_ZN ×2
_MG ×2
Waters ×439
* Residue conservation analysis
PDB id:
Name: Alkaline phosphatase
Title: Alkaline phosphatase (n51mg)
Structure: Alkaline phosphatase. Chain: a, b. Engineered: yes. Mutation: yes
Source: Escherichia coli. Organism_taxid: 562. Strain: ek1734. Gene: phoa. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PQS)
2.14Å     R-factor:   0.193     R-free:   0.241
Authors: T.T.Tibbitts,J.E.Murphy,E.R.Kantrowitz
Key ref:
T.T.Tibbitts et al. (1996). Kinetic and structural consequences of replacing the aspartate bridge by asparagine in the catalytic metal triad of Escherichia coli alkaline phosphatase. J Mol Biol, 257, 700-715. PubMed id: 8648634 DOI: 10.1006/jmbi.1996.0195
03-Feb-96     Release date:   11-Jul-96    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
P00634  (PPB_ECOLI) -  Alkaline phosphatase
471 a.a.
446 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.  - Alkaline phosphatase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: A phosphate monoester + H2O = an alcohol + phosphate
phosphate monoester
+ H(2)O
= alcohol
Bound ligand (Het Group name = PO4)
corresponds exactly
      Cofactor: Mg(2+); Zn(2+)
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  


DOI no: 10.1006/jmbi.1996.0195 J Mol Biol 257:700-715 (1996)
PubMed id: 8648634  
Kinetic and structural consequences of replacing the aspartate bridge by asparagine in the catalytic metal triad of Escherichia coli alkaline phosphatase.
T.T.Tibbitts, J.E.Murphy, E.R.Kantrowitz.
In each subunit of the homodimeric enzyme Escherichia coli alkaline phosphatase, two of the three metal cofactors Zn2+ and Mg2+, are bound by an aspartate side-chain at position 51. Using site-specific mutagenesis, Asp51 was mutated both to alanine and to asparagine to produce the D51A and D51N enzymes, respectively. Over the range of pH values examined, the D51A enzyme did not catalyze phosphate ester hydrolysis above non-enzymic levels and was not activated by the addition of millimolar excess Zn2+ or Mg2+. Replacement of Asp51 by asparagine, however, resulted in a mutant enzyme with reduced activity and a higher pH optimum, compared with the wild-type enzyme. At pH 8.0 the D51N enzyme showed about 1% of the activity of the wild-type enzyme, and as the pH was raised to 9.2, the activity of the D51N enzyme increased to about 10% of the value for the wild-type enzyme. Upon the addition of excess Mg2+ at pH 9.2, the D51N enzyme was activated in a time-dependent fashion to nearly the same level as the wild-type enzyme. The affinity for phosphate of the D51N enzyme decreased tenfold as the concentration of Mg2+ increased. Under optimal conditions, the k(cat)/K(m) ratio for the D51N enzyme indicated that it was 87% as efficient as the wild-type enzyme. To investigate the molecular basis for the observed kinetic differences, X-ray data were collected for the D51N enzyme to 2.3 angstroms resolution at pH 7.5, and then to 2.1 angstroms resolution at pH 9.2 with 20 mM MgCl2. The two structures were then refined. The low magnesium, low pH D51N structure showed that the third metal site was unoccupied, apparently blocked by the amide group of Asn51. At this pH the phosphate anion was bound via one oxygen atom, between the zinc cations at the first and second metal sites, which strongly resembled the arrangement previously determined for the D153H enzyme at pH 7.5. In the high magnesium, high pH D51N structure, the third metal site was also vacant, but the phosphate anion bound closer to the surface of the enzyme, coordinated to the first metal site alone. Electron density difference maps provide evidence that magnesium activates the D51N enzyme by replacing zinc at the second metal site.
  Selected figure(s)  
Figure 7.
Figure 7. Average hydrogen bond and metal-ligand distances in the active sites of the N51Zn structure. Two of the metal sites (M1 and M2) are occupied by zinc atoms (Zn) and the third metal site is unoccupied.
Figure 11.
Figure 11. Average hydrogen bond and metal-ligand distances in the active sites of the H153Zn structure. In this structure (unlike the N51Zn and N51Mg structures, compare Figures 7 and 8) all three metal sites are occupied by Zn 2+ .
  The above figures are reprinted by permission from Elsevier: J Mol Biol (1996, 257, 700-715) copyright 1996.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
  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
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
15725663 Y.Suzuki, Y.Mizutani, T.Tsuji, N.Ohtani, K.Takano, M.Haruki, M.Morikawa, and S.Kanaya (2005).
Gene cloning, overproduction, and characterization of thermolabile alkaline phosphatase from a psychrotrophic bacterium.
  Biosci Biotechnol Biochem, 69, 364-373.  
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.  
14764088 R.Banerjee, D.Y.Dubois, J.Gauthier, S.X.Lin, S.Roy, and J.Lapointe (2004).
The zinc-binding site of a class I aminoacyl-tRNA synthetase is a SWIM domain that modulates amino acid binding via the tRNA acceptor arm.
  Eur J Biochem, 271, 724-733.  
11937510 A.Kozlenkov, T.Manes, M.F.Hoylaerts, and J.L.Millán (2002).
Function assignment to conserved residues in mammalian alkaline phosphatases.
  J Biol Chem, 277, 22992-22999.  
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.  
10551856 J.C.Taylor, and G.D.Markham (1999).
The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.
  J Biol Chem, 274, 32909-32914.  
10584076 M.Bortolato, F.Besson, and B.Roux (1999).
Role of metal ions on the secondary and quaternary structure of alkaline phosphatase from bovine intestinal mucosa.
  Proteins, 37, 310-318.  
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.