PDBsum entry: 1elz

Hydrolase

PDB id: 1elz

Contents

Protein chains

449 a.a. *

Ligands

PO4 ×2

Metals

_ZN ×4

_MG ×2

Waters ×676

* Residue conservation analysis

PDB id:

1elz

Name:

Hydrolase

Title:

E. Coli alkaline phosphatase mutant (s102g)

Structure:

Alkaline phosphatase. Chain: a, b. Engineered: yes. Mutation: yes. Other_details: enzyme inhibited with phosphate

Source:

Escherichia coli. Organism_taxid: 562. Strain: sm547. Gene: phoa. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.80Å R-factor: 0.138 R-free: 0.173

Authors:

B.Stec,M.Hehir,C.Brennan,M.Nolte,E.R.Kantrowitz

Key ref:

B.Stec et al. (1998). Kinetic and X-ray structural studies of three mutant E. coli alkaline phosphatases: insights into the catalytic mechanism without the nucleophile Ser102. J Mol Biol, 277, 647-662. PubMed id: 9533886 DOI: 10.1006/jmbi.1998.1635

Date:

10-Feb-98 Release date: 27-May-98

Protein chains

P00634  (PPB_ECOLI) -  Alkaline phosphatase

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

phosphate monoester + H(2)O = alcohol + phosphate (Bound ligand (Het Group name = PO4) corresponds exactly)

• 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: 10.1006/jmbi.1998.1635

J Mol Biol 277:647-662 (1998)

PubMed id: 9533886

Kinetic and X-ray structural studies of three mutant E. coli alkaline phosphatases: insights into the catalytic mechanism without the nucleophile Ser102.

B.Stec, M.J.Hehir, C.Brennan, M.Nolte, E.R.Kantrowitz.

ABSTRACT

Escherichia coli alkaline phosphatase (EC 3.1.3.1) is a non-specific phosphomonoesterase that catalyzes the hydrolysis reaction via a phosphoseryl intermediate to produce inorganic phosphate and the corresponding alcohol. We investigated the nature of the primary nucleophile, fulfilled by the deprotonated Ser102, in the catalytic mechanism by mutating this residue to glycine, alanine and cysteine. The efficiencies of the S102G, S102A and S102C enzymes were 6 x 10(5)-fold, 10(5)-fold and 10(4)-fold lower than the wild-type enzyme, respectively, as measured by the kcat/Km ratio, still substantially higher than the non-catalyzed reaction. In order to investigate the structural details of the altered active site, the enzymes were crystallized and their structures determined. The enzymes crystallized in a new crystal form corresponding to the space group P6322. Each structure has phosphate at each active site and shows little departure from the wild-type model. For the S102G and S102A enzymes, the phosphate occupies the same position as in the wild-type enzyme, while in the S102C enzyme it is displaced by 2.5 A. This kinetic and structural study suggests an explanation for differences in catalytic efficiency of the mutant enzymes and provides a means to study the nature and strength of different nucleophiles in the same environment. The analysis of these results provides insight into the mechanisms of other classes of phosphatases that do not utilize a serine nucleophile.

Selected figure(s)

Figure 2.
Figure 2. The active site of the wild-type E. coli alkaline phosphatase. Shown are Zn[1], Zn[2], Mg, phosphate (P[i]) and the side-chain ligands. Also shown is Ser102, which is phosphorylated during the reaction, and Arg166, which interacts with the phosphate.

Figure 4.
Figure 4. Stereoview of the (2F[o]−F[c]) electron density map (contoured at 1.3σ) of the active site in the S102A mutant structure. The electron density and the side-chain for one of the Zn[2]ligands (His370) are omitted for clarity.

The above figures are reprinted by permission from Elsevier: J Mol Biol (1998, 277, 647-662) copyright 1998.

Figures were selected by an automated process.