PDBsum entry 2g9y

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Hydrolase PDB id
Protein chains
449 a.a. *
PO4 ×2
SO4 ×2
_ZN ×4
_MG ×2
Waters ×552
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Structure of s102t e. Coli alkaline phosphatase in presence of phosphate at 2.00 a resolution
Structure: Alkaline phosphatase. Chain: a, b. Synonym: apase. Engineered: yes. Mutation: yes
Source: Escherichia coli. Organism_taxid: 562. Gene: phoa. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PQS)
2.00Å     R-factor:   0.205     R-free:   0.242
Authors: J.Wang,E.R.Kantrowitz
Key ref:
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. PubMed id: 17008720 DOI: 10.1110/ps.062351506
07-Mar-06     Release date:   10-Oct-06    
Go to PROCHECK summary

Protein chains
No UniProt id for this chain
Struc: 449 a.a.
Key:    Secondary structure  CATH domain

 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: 10.1110/ps.062351506 Protein Sci 15:2395-2401 (2006)
PubMed id: 17008720  
Trapping the tetrahedral intermediate in the alkaline phosphatase reaction by substitution of the active site serine with threonine.
J.Wang, E.R.Kantrowitz.
We report here the construction of a mutant version of Escherichia coli alkaline phosphatase (AP) in which the active site Ser was replaced by Thr (S102T), in order to investigate whether the enzyme can utilize Thr as the nucleophile and whether the rates of the critical steps in the mechanism are altered by the substitution. The mutant AP with Thr at position 102 exhibited an approximately 4000-fold decrease in k(cat) along with a small decrease in Km. The decrease in catalytic efficiency of approximately 2000-fold was a much smaller drop than that observed when Ala or Gly were substituted at position 102. The mechanism by which Thr can substitute for Ser in AP was further investigated by determining the X-ray structure of the S102T enzyme in the presence of the Pi (S102T_Pi), and after soaking the crystals with substrate (S102T_sub). In the S102T_Pi structure, the Pi was coordinated differently with its position shifted by 1.3 A compared to the structure of the wild-type enzyme in the presence of Pi. In the S102T_sub structure, a covalent Thr-Pi intermediate was observed, instead of the expected bound substrate. The stereochemistry of the phosphorus in the S102T_sub structure was inverted compared to the stereochemistry in the wild-type structure, as would be expected after the first step of a double in-line displacement mechanism. We conclude that the S102T mutation resulted in a shift in the rate-determining step in the mechanism allowing us to trap the covalent intermediate of the reaction in the crystal.
  Selected figure(s)  
  The above figure is reprinted by permission from the Protein Society: Protein Sci (2006, 15, 2395-2401) copyright 2006.  
  Figure was selected by an automated process.