PDBsum entry 1alh

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Hydrolase (phosphoric monoester) PDB id
Protein chains
446 a.a. *
PO4 ×2
SO4 ×2
_ZN ×2
Waters ×888
* Residue conservation analysis
PDB id:
Name: Hydrolase (phosphoric monoester)
Title: Kinetics and crystal structure of a mutant e. Coli alkaline phosphatase (asp-369-->asn): a mechanism involving one zinc per active site
Structure: Alkaline phosphatase. Chain: a, b. Engineered: yes
Source: Escherichia coli. Organism_taxid: 562. Cell_line: sm547. Gene: phoa. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PQS)
2.50Å     R-factor:   0.189    
Authors: T.T.Tibbitts,X.Xu,E.R.Kantrowitz
Key ref:
T.T.Tibbitts et al. (1994). Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp-369-->Asn): a mechanism involving one zinc per active site. Protein Sci, 3, 2005-2014. PubMed id: 7703848 DOI: 10.1002/pro.5560031113
23-Aug-94     Release date:   27-Feb-95    
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 2 residue positions (black crosses)

 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: 10.1002/pro.5560031113 Protein Sci 3:2005-2014 (1994)
PubMed id: 7703848  
Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp-369-->Asn): a mechanism involving one zinc per active site.
T.T.Tibbitts, X.Xu, E.R.Kantrowitz.
Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme. Thus the kcat/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 A resolution with an R-factor of 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.
  Selected figure(s)  
Figure 6.
Fig. 6. Stereo pair showing the electron density of the anion binding site ound in D369N alkaline phosphatase. The ligand at this site, which may be ei- phosphate or sulfate, was modeled assulfatedurigthe refinement. Fig- 6,7,and 8 were produced sing the program SETOR (Evans, 1993).
Figure 8.
Fig. 8. pair comparing the posi- tions of side chains, metals, andphos- phate in the active sites of the D369N enzyme (thick lines) and the wild-type enzyme (thin lines). At the I site, zinc is bound close to he same location in both enzymes (crosses), but phosphate (PO,) is more exposed to the surface in the D369N structure. At the M2 site, zinc is bound n the wild-type (cross) but not theutant enzyme. This permits SI02 to move slightly closer to H370 and the asparagineintroduce at position 369 (D369N). The M3 site normally contains Mgz+ (cross) with 3 water molecules (not shown) in the wild-type enzyme; in the mutant enzyme this space is partially filled y 1 water molecule (not shown), the carboxylate side chain of D5 1, and the#-aminogroup of K328. The 2 sets of atomiccordinates were aligned using Quanta to minimize the RMSD of the Cor atomsbefore making this omparison.
  The above figures are reprinted from an Open Access publication published by the Protein Society: Protein Sci (1994, 3, 2005-2014) copyright 1994.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19349157 W.Li, L.Bi, W.Wang, Y.Li, Y.Zhou, H.Wei, T.Jiang, L.Bai, Y.Chen, Z.Zhang, X.Yuan, J.Xiao, and X.E.Zhang (2009).
Development of a universal phosphorylated peptide-binding protein for simultaneous assay of kinases.
  Biosens Bioelectron, 24, 2871-2877.
PDB code: 3dpc
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.  
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.  
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.  
  8520475 L.Ma, T.T.Tibbitts, and E.R.Kantrowitz (1995).
Escherichia coli alkaline phosphatase: X-ray structural studies of a mutant enzyme (His-412-->Asn) at one of the catalytically important zinc binding sites.
  Protein Sci, 4, 1498-1506.
PDB codes: 1ali 1alj
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