Functional importance of calcium binding sites in outer membrane phospholipase A

Biochim Biophys Acta. 2002 Apr 12;1561(2):230-7. doi: 10.1016/s0005-2736(02)00351-6.

Abstract

Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that hydrolyses phospholipids requiring Ca(2+) as cofactor. In vitro studies have shown that OMPLA is only active as a dimer. The structures of monomeric and dimeric OMPLA provided possible clues to the activation process. In the inhibited dimeric species calcium ions are located at the dimer interface ideally suited to stabilise the oxyanion intermediates formed during catalysis. The side chain hydroxyl function of Ser152 is one of the ligands of this interfacial calcium. In the crystal structure of monomeric OMPLA the interfacial calcium site is lacking, but calcium was found to bind at a site involving the carboxylates of Asp149 and Asp184. In the current study the relevance of the identified calcium sites has been studied by site-directed mutagenesis. The Ser152Asn variant confirmed the importance of the interfacial calcium site for catalysis, and also demonstrated that this site is essentially involved in the dimerisation process. Replacements of the ligands in monomeric OMPLA, i.e. Asp149Asn, Asp149Ala and Asp184Asn, only showed minor effects on catalytic activity and dimerisation. A stronger effect observed for the variant Asp184Ala was explained by the proximity of Asp184 to the catalytically important Ser152 residue. We propose that Asp149 and Asp184 provide an electronegative funnel that may facilitate Ca(2+) transfer to the interfacial calcium site.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Outer Membrane Proteins / chemistry
  • Bacterial Outer Membrane Proteins / genetics
  • Bacterial Outer Membrane Proteins / metabolism*
  • Binding Sites
  • Calcium / chemistry
  • Calcium / metabolism*
  • Chromatography, Gel
  • Dimerization
  • Kinetics
  • Mutagenesis, Site-Directed
  • Mutation
  • Phospholipases A / chemistry
  • Phospholipases A / genetics
  • Phospholipases A / metabolism*
  • Phospholipases A1

Substances

  • Bacterial Outer Membrane Proteins
  • Phospholipases A
  • Phospholipases A1
  • outer membrane phospholipase A
  • Calcium