title: Activities of native and tyrosine-69 mutant phospholipases A2 on phospholipid analogues. A reevaluation of the minimal substrate requirements.
abstract: The role of Tyr-69 of porcine pancreatic phospholipase A2 in substrate binding was studied with the help of proteins modified by site-directed mutagenesis and phospholipid analogues with a changed head-group geometry. Two mutants were used containing Phe and Lys, respectively, at position 69. Modifications in the phospholipids included introduction of a sulfur at the phosphorus (thionophospholipids), removal of the negative charge at phosphorus (phosphatidic acid dimethyl ester), and reduction (phosphonolipids) or extension (diacylbutanetriol choline phosphate) of the distance between the phosphorus and the acyl ester bond. Replacement of Tyr-69 by Lys reduces enzymatic activity, but the mutant enzyme retains both the stereospecificity and positional specificity of native phospholipase A2. The Phe-69 mutant not only hydrolyzes the Rp isomer of thionophospholipids more efficiently than the wild-type enzyme, but the Sp thiono isomer is hydrolyzed too, although at a low (approximately 4%) rate. Phosphonolipids are hydrolyzed by native phospholipase A2 about 7 times more slowly than natural phospholipids, with retention of positional specificity and a (partial) loss of stereospecificity. The dimethyl ester of phosphatidic acid is degraded efficiently in a calcium-dependent and positional-specific way by native phospholipase A2 and by the mutants, indicating that a negative charge at phosphorus is not an absolute substrate requirement. The activities on the phosphatidic acid dimethyl ester of native enzyme and the Lys-69 mutant are lower than those on the corresponding lecithin, in contrast to the Phe-69 mutant, which has equal activities on both substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Utrecht, The Netherlands.
authors.forename: O P
authors.initials: OP
authors.lastname: Kuipers
authors.forename: N
authors.initials: N
authors.lastname: Dekker
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; D001483:Base Sequence; D004952:Esters / Q000378:metabolism; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010179:Pancreas / Q000201:enzymology; D010649:Phenylalanine / Q000235:genetics; D010710:Phosphates / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine; D014443:Tyrosine / Q000235:genetics*
chemical_list: D004952:Esters; D010710:Phosphates; D010713:Phosphatidylcholines; D010743:Phospholipids; D014443:Tyrosine; D010649:Phenylalanine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine