title: Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2.
abstract: In the active centre of pancreatic phospholipase A2 His48 is at hydrogen-bonding distance to Asp99. This Asp-His couple is assumed to act together with a water molecule as a catalytic triad. Asp99 is also linked via an extended hydrogen bonding system to the side chains of Tyr52 and Tyr73. To probe the function of the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipase A2, the Asp99 residue was replaced by Asn, and each of the two tyrosines was separately replaced by either a Phe or a Gln. The catalytic and binding properties of the Phe52 and Phe73 mutants did not change significantly relative to the wild-type enzyme. This rules out the possibility that either one of the two Tyr residues in the wild-type enzyme can function as an acyl acceptor or proton donor in catalysis. The Gln73 mutant could not be obtained in any significant amounts probably due to incorrect folding. The Gln52 mutant was isolated in low yield. This mutant showed a large decrease in catalytic activity while its substrate binding was nearly unchanged. The results suggest a structural role rather than a catalytic function of Tyr52 and Tyr73. Substitution of asparagine for aspartate hardly affects the binding constants for both monomeric and micellar substrate analogues. Kinetic characterization revealed that the Asn99 mutant has retained no less than 65% of its enzymatic activity on the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine, probably due to the fact that during hydrolysis of monomeric substrate by phospholipase A2 proton transfer is not the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, State University of Utrecht, The Netherlands.
authors.forename: O P
authors.initials: OP
authors.lastname: Kuipers
authors.forename: P A
authors.initials: PA
authors.lastname: Franken
authors.forename: R
authors.initials: R
authors.lastname: Hendriks
authors.forename: H M
authors.initials: HM
authors.lastname: Verheij
authors.forename: G H
authors.initials: GH
authors.lastname: de Haas
mesh_terms: D001216:Asparagine / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054467:Phospholipases A2; D015202:Protein Engineering; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D014443:Tyrosine; D001216:Asparagine; D010741:Phospholipases A; D054467:Phospholipases A2