title: Changes in activity of porcine phospholipase A2 brought about by charge engineering of a major structural element to alter stability.
abstract: We have modified the stability of porcine phospholipase A2 by charge engineering. The mutations are situated at the N-terminal of a major helix and are N89D and N89D/E92Q. This engineering has significantly altered the activity of the enzyme to aggregated and monomeric substrates. A N89D/E92K mutant is more stable but considerably less active than wild type. An N89D mutant is more stable and of similar activity to wild type. The substantial change in activity may be due to direct interaction of residue 92 with aggregated substrate or may be via second calcium binding. Second calcium binding may be more probable as activity against monomers is also affected. Additional calcium binding may therefore be an important way of manipulating the activity of phospholipase A2.
authors.affiliation: Protein Engineering Department, AFRC Institute of Food Research, Shinfield, Reading, UK.
authors.forename: P W
authors.initials: PW
authors.lastname: Goodenough
authors.forename: K M
authors.initials: KM
authors.lastname: Bhat
authors.forename: M E
authors.initials: ME
authors.lastname: Collins
authors.forename: B N
authors.initials: BN
authors.lastname: Perry
authors.forename: R W
authors.initials: RW
authors.lastname: Pickersgill
authors.forename: I G
authors.initials: IG
authors.lastname: Sumner
authors.forename: J
authors.initials: J
authors.lastname: Warwicker
authors.forename: G H
authors.initials: GH
authors.lastname: de Haas
authors.forename: H M
authors.initials: HM
authors.lastname: Verheij
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics; D054467:Phospholipases A2; D011489:Protein Denaturation; D015202:Protein Engineering; D013552:Swine / Q000235:genetics
chemical_list: D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium