PDBsum entry 1bwp

Hydrolase

PDB id: 1bwp

Contents

Protein chain

212 a.a. *

Waters ×145

* Residue conservation analysis

PDB id:

1bwp

Name:

Hydrolase

Title:

Probing the substrate specificity of the intracellular brain platelet- activating factor acetylhydrolase

Structure:

Platelet-activating factor acetylhydrolase. Chain: a. Synonym: pafah. Engineered: yes. Mutation: yes

Source:

Bos taurus. Cattle. Organism_taxid: 9913. Organ: brain. Tissue: brain. Cellular_location: cytoplasm. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Biol. unit:

Homo-Dimer (from PDB file)

Resolution:

2.10Å R-factor: 0.209 R-free: 0.252

Authors:

Y.S.Ho,P.J.Sheffield,J.Masuyama,H.Arai,J.Li,J.Aoki,K.Inoue, U.Derewenda,Z.Derewenda

Key ref:

Y.S.Ho et al. (1999). Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase. Protein Eng, 12, 693-700. PubMed id: 10469831

Date:

27-Sep-98 Release date: 18-May-99

Protein chain

Q29460  (PA1B3_BOVIN) -  Platelet-activating factor acetylhydrolase IB subunit alpha1 from Bos taurus

Seq: 232 a.a.

Struc: 212 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.1.1.47 - 1-alkyl-2-acetylglycerophosphocholine esterase.
• Reaction: a 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine + H2O = a 1-O-alkyl- sn-glycero-3-phosphocholine + acetate + H⁺

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

Key reference

Protein Eng 12:693-700 (1999)

PubMed id: 10469831

Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.

Y.S.Ho, P.J.Sheffield, J.Masuyama, H.Arai, J.Li, J.Aoki, K.Inoue, U.Derewenda, Z.S.Derewenda.

ABSTRACT

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.