PDBsum entry 1hqa

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Hydrolase (alkaline phosphatase) PDB id
Protein chains
446 a.a. *
_ZN ×6
Waters ×393
* Residue conservation analysis
PDB id:
Name: Hydrolase (alkaline phosphatase)
Title: Alkaline phosphatase (h412q)
Structure: Alkaline phosphatase. Chain: a, b. Engineered: yes. Mutation: yes. Other_details: crystallized from 55% saturating (nh4)2so4, 100 mm tris, 10 mm mgcl2 10 mm zncl2, 2 mm nah2po4, ph 7.5
Source: Escherichia coli. Organism_taxid: 562. Strain: ek1685. Gene: phoa. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PQS)
2.25Å     R-factor:   0.161    
Authors: L.Ma,E.R.Kantrowitz
Key ref:
L.Ma and E.R.Kantrowitz (1996). Kinetic and X-ray structural studies of a mutant Escherichia coli alkaline phosphatase (His-412-->Gln) at one of the zinc binding sites. Biochemistry, 35, 2394-2402. PubMed id: 8652582 DOI: 10.1021/bi9523421
30-Nov-95     Release date:   08-Mar-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
+ phosphate
      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: 10.1021/bi9523421 Biochemistry 35:2394-2402 (1996)
PubMed id: 8652582  
Kinetic and X-ray structural studies of a mutant Escherichia coli alkaline phosphatase (His-412-->Gln) at one of the zinc binding sites.
L.Ma, E.R.Kantrowitz.
Site-specific mutagenesis has been used to replace His-412 with glutamine in Escherichia coli alkaline phosphatase. In the wild-type enzyme His-412 is a direct ligand to one of the catalytically important zinc atoms (Zn1) in the active site. The mutant enzyme (H412Q) exhibited about the same k(cat), but a 50-fold increase in K(m) compared to the corresponding kinetic parameters for the wild-type enzyme. Furthermore, the H412Q enzyme had a lower zinc content than the wild-type enzyme. In contrast to the wild-type enzyme, Tris was less effective in the transferase reaction and dramatically inhibited the hydrolysis reaction of the H412Q enzyme. The addition of zinc to the mutant enzyme increased the k(cat) value above that of the wild-type enzyme, partially restored the weak substrate and phosphate binding, and also alleviated the inhibition by Tris. The structure of the H412Q enzyme was also determined by X-ray crystallography. The overall structure of the H412Q enzyme was very similar to that of the wild-type enzyme; the only alpha-carbon displacements over 1 angstrom were observed near the mutation site. In the H412Q structure no phosphate was bound in the active site of the enzyme; however, two water molecules were observed where phosphate normally binds in the wild-type enzyme. Close examination of the active site of the H412Q structure revealed structural changes in Ser-102 as well as at the mutation site. For example, the carbonyl oxygen of the side chain of Gln-412 rotated away from the position of His-412 in the wild-type structure, although too far away (3.2 angstroms) to coordinate to Zn1. Studies on the H412Q enzyme, and a comparison of the H412Q and H412N structures, suggest that the structure and electostatics of the imidazole ring of histidine are critical for its function as a zinc ligand in alkaline phosphatase.

Literature references that cite this PDB file's key reference

  PubMed id Reference
15576371 S.R.Shouldice, D.E.McRee, D.R.Dougan, L.W.Tari, and A.B.Schryvers (2005).
Novel anion-independent iron coordination by members of a third class of bacterial periplasmic ferric ion-binding proteins.
  J Biol Chem, 280, 5820-5827.
PDB codes: 1xvx 1xvy
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.  
11148046 J.E.Jackman, C.R.Raetz, and C.A.Fierke (2001).
Site-directed mutagenesis of the bacterial metalloamidase UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC). Identification of the zinc binding site.
  Biochemistry, 40, 514-523.  
10353848 H.Teng, and C.Grubmeyer (1999).
Mutagenesis of histidinol dehydrogenase reveals roles for conserved histidine residues.
  Biochemistry, 38, 7363-7371.  
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.  
8841144 S.F.Martin, M.R.Spaller, and P.J.Hergenrother (1996).
Expression and site-directed mutagenesis of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus: probing the role of the active site Glu146.
  Biochemistry, 35, 12970-12977.  
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