title: Evidence for the involvement of tyrosine-69 in the control of stereospecificity of porcine pancreatic phospholipase A2.
abstract: We have studied the role of Tyr-69 of porcine pancreatic phospholipase A2 in catalysis and substrate binding, using site-directed mutagenesis. A mutant was constructed containing Phe at position 69. Kinetic characterization revealed that the Phe-69 mutant has retained enzymatic activity on monomeric and micellar substrates, and that the mutation has only minor effects on kcat and Km. This shows that Tyr-69 plays no role in the true catalytic events during substrate hydrolysis. In contrast, the mutation has a profound influence on the stereospecificity of the enzyme. Whereas the wild-type phospholipase A2 is only able to catalyse the degradation of sn-3 phospholipids, the Phe-69 mutant hydrolyses both the sn-3 isomers and, at a low (1-2%) rate, the sn-1 isomers. Despite the fact that the stereospecificity of the mutant phospholipase has been altered, Phe-69 phospholipase still requires Ca2+ ions as a cofactor and also retains its specificity for the sn-2 ester bond. Our data suggest that in porcine pancreatic phospholipase A2 the hydroxyl group of Tyr-69 serves to fix and orient the phosphate group of phospholipid monomers by hydrogen bonding. Because no such interaction can occur between the Phe-69 side-chain and the phosphate moiety of the substrate monomer, the mutant enzyme loses part of its stereospecificity but not its positional specificity.
authors.affiliation: Department of Biochemistry, State University of Utrecht, The Netherlands.
authors.forename: O P
authors.initials: OP
authors.lastname: Kuipers
authors.forename: R
authors.initials: R
authors.lastname: Dijkman
authors.forename: C E
authors.initials: CE
authors.lastname: Pals
authors.forename: H M
authors.initials: HM
authors.lastname: Verheij
authors.forename: G H
authors.initials: GH
authors.lastname: de Haas
mesh_terms: D000818:Animals; D010179:Pancreas / Q000201:enzymology*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D015202:Protein Engineering; D013379:Substrate Specificity; D013552:Swine; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D014443:Tyrosine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2