PDBsum entry 2ga3

Hydrolase

PDB id: 2ga3

Contents

Protein chains

449 a.a. *

Ligands

SO4 ×2

Metals

_ZN ×4

_MG ×2

Waters ×495

* Residue conservation analysis

PDB id:

2ga3

Name:

Hydrolase

Title:

Structure of s102t e. Coli alkaline phosphatase-phosphate intermediate at 2.20a 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)

Resolution:

2.20Å R-factor: 0.192 R-free: 0.229

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

Date:

07-Mar-06 Release date: 10-Oct-06

Protein chains

P00634  (PPB_ECOLI) -  Alkaline phosphatase from Escherichia coli (strain K12)

Seq: 471 a.a.

Struc: 449 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.1.3.1 - alkaline phosphatase.
• Reaction: a phosphate monoester + H2O = an alcohol + phosphate
• Cofactor: Mg(2+); Zn(2+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

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.

ABSTRACT

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.