Evidence for the involvement of tyrosine-69 in the control of stereospecificity of porcine pancreatic phospholipase A2 |
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Authors: | Kuipers, Oscar P. Dijkman, Ruud Pals, Cornelieke E.G.M. Verheij, Hubertus M. Haas, Gerard H.de |
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Affiliation: | Department of Biochemistry, State University of Utrecht Padualaan 8, PO Box 80.054, NL-3508 TB Utrecht, The Netherlands |
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Abstract: | We have studied the role of Tyr-69 of porcine pancreatic phospholipaseA2 in catalysis and substrate binding, using site-directed mutagenesis.A mutant was constructed containing Phe at position 69. Kineticcharacterization revealed that the Phe-69 mutant has retainedenzymatic activity on monomeric and micellar substrates, andthat the mutation has only minor effects on kcat and Km. Thisshows that Tyr-69 plays no role in the true catalytic eventsduring substrate hydrolysis. In contrast, the mutation has aprofound influence on the stereospecificity of the enzyme. Whereasthe wild-type phospholipase A2 is only able to catalyse thedegradation of sn-3 phospholipids, the Phe-69 mutant hydrolysesboth the sn-3 isomers and, at a low (12%) rate, the sn-1isomers. Despite the fact that the stereospecificity of themutant phospholipase has been altered, Phe-69 phospholipasestill requires Ca2+ ions as a cofactor and also retains itsspecificity for the sn-2 ester bond. Our data suggest that inporcine pancreatic phospholipase A2 the hydroxyl group of Tyr-69serves to fix and orient the phosphate group of phospholipidmonomers by hydrogen bonding. Because no such interaction canoccur between the Phe-69 side-chain and the phosphate moietyof the substrate monomer, the mutant enzyme loses part of itsstereospecificity but not its positional specificity. |
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Keywords: | phospholipase A2/ site-directed mutagenesis/ stereospecificity/ substrate specificity/ Tyr-69 |
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