PDBsum entry: 1ajb

Non specific mono-esterase

PDB id: 1ajb

Contents

Protein chains

449 a.a. *

Ligands

SO4 ×2

Metals

_ZN ×4

_MG ×2

Waters ×167

* Residue conservation analysis

PDB id:

1ajb

Name:

Non specific mono-esterase

Title:

Three-dimensional structure of the d153g mutant of e. Coli alkaline phosphatase: a mutant with weaker magnesium binding and increased catalytic activity

Structure:

Alkaline phosphatase. Chain: a, b. Engineered: yes. Mutation: yes. Other_details: mutant with 4-fold increased activity and weaker mg binding

Source:

Escherichia coli. Organism_taxid: 562. Gene: phoa. Expressed in: escherichia coli. Expression_system_taxid: 562. Restriction fragment. Other_details: lac promoter. For more information about the expression system consult mandecki et al. Gene 94, 103- 107. (1990).

Biol. unit:

Dimer (from PQS)

Resolution:

2.50Å R-factor: 0.162

Authors:

C.G.Dealwis,L.Chen,C.Abad-Zapatero

Key ref:

C.G.Dealwis et al. (1995). 3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity. Protein Eng, 8, 865-871. PubMed id: 8746724 DOI: 10.1093/protein/8.9.865

Date:

19-Aug-95 Release date: 14-Nov-95

Protein chains

P00634  (PPB_ECOLI) -  Alkaline phosphatase

Seq: 471 a.a.

Struc: 449 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.1.3.1 - Alkaline phosphatase.
• Reaction: A phosphate monoester + H₂O = an alcohol + phosphate
• Cofactor: Magnesium; Zinc

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

 Gene Ontology (GO) functional annotation 

• Cellular component: periplasmic space (1 term)
• Biological process: metabolic process (2 terms)
• Biochemical function: catalytic activity (10 terms)

reference

DOI no: 10.1093/protein/8.9.865

Protein Eng 8:865-871 (1995)

PubMed id: 8746724

3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity.

C.G.Dealwis, L.Chen, C.Brennan, W.Mandecki, C.Abad-Zapatero.

ABSTRACT

The substitution of aspartate at position 153 in Escherichia coli alkaline phosphatase by glycine results in a mutant enzyme with 5-fold higher catalytic activity (kcat) but no change in Km at pH 8.0 in 50 mM Tris-HCl. The increased kcat is achieved by a faster release of the phosphate product as a result of the lower phosphate affinity. The mutation also affects Mg2+ binding, resulting in an enzyme with lower metal affinity. The 3-D X-ray structure of the D153G mutant has been refined at 2.5 A to a crystallographic R-factor of 16.2%. An analysis of this structure has revealed that the decreased phosphate affinity is caused by an apparent increase in flexibility of the guanidinium side chain of Arg166 involved in phosphate binding. The mutation of Asp153 to Gly also affects the position of the water ligands of Mg2+, and the loop Gln152-Thr155 is shifted by 0.3 A away from the active site. The weaker Mg2+ binding of the mutant compared with the wild type is caused by an altered coordination sphere in the proximity of the Mg2+ ion, and also by the loss of an electrostatic interaction (Mg2+.COO-Asp153) in the mutant. Its ligands W454 and W455 and hydroxyl of Thr155, involved in the octahedral coordination of the Mg2+ ion, are further apart in the mutant compared with the wild type.