title: Secretory phospholipase A2 hydrolysis of phospholipid analogues is dependent on water accessibility to the active site.
abstract: A new and unnatural type of phospholipids with the head group attached to the 2-position of the glycerol backbone has been synthesized and shown to be a good substrate for secretory phospholipase A2 (sPLA2). To investigate the unexpected sPLA2 activity, we have compared three different phospholipids by using fluorescence techniques and HPLC, namely: (R)-1,2-dipalmitoyl-glycero-3-phosphocholine (hereafter referred to as 1R), (R)-1-O-hexadecyl-2-palmitoyl-glycero-3-phoshocholine (2R), and (S)-1-O-hexadecyl-3-palmitoyl-glycero-2-phosphocholine (3S). Furthermore, to understand the underlying mechanisms for the observed differences, we have performed molecular dynamics simulations to clarify on a structural level the substrate specificity of sPLA2 toward phospholipid analogues with their head groups in the 2-position of the glycerol backbone. We have studied the lipids above 1R, 2R, and 3S as well as their enantiomers 1S, 2S, and 3R. In the simulations of sPLA2-1S and sPLA2-3R, structural distortion in the binding cleft induced by the phospholipids showed that these are not substrates for sPLA2. In the case of the phospholipids 1R, 2R, and 3S, our simulations revealed that the difference observed experimentally in sPLA2 activity might be caused by reduced access of water molecules to the active site. We have monitored the number of water molecules that enter the active site region for the different sPLA2-phospholipid complexes and found that the probability of a water molecule reaching the correct position such that hydrolysis can occur is reduced for the unnatural lipids. The relative water count follows 1R > 2R > 3S. This is in good agreement with experimental data that indicate the same trend for sPLA2 activity: 1R > 2R > 3S.
authors.affiliation: Department of Chemistry, MEMPHYS-Center for Biomembrane Physics, Technical University of Denmark, DK-2800 Lyngby, Denmark.
authors.forename: Günther H
authors.initials: GH
authors.lastname: Peters
authors.forename: Martin S
authors.initials: MS
authors.lastname: Møller
authors.forename: Kent
authors.initials: K
authors.lastname: Jørgensen
authors.forename: Petra
authors.initials: P
authors.lastname: Rönnholm
authors.forename: Mette
authors.initials: M
authors.lastname: Mikkelsen
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Andresen
mesh_terms: D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry; D017836:Agkistrodon; D000818:Animals; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D008081:Liposomes / Q000737:chemistry; D008958:Models, Molecular; D008968:Molecular Conformation; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D010743:Phospholipids / Q000737:chemistry*; D014757:Viper Venoms / Q000201:enzymology; D014867:Water / Q000737:chemistry
chemical_list: D007202:Indicators and Reagents; D008081:Liposomes; D010743:Phospholipids; D014757:Viper Venoms; D014867:Water; D015060:1,2-Dipalmitoylphosphatidylcholine; D010741:Phospholipases A; D054467:Phospholipases A2