Evidence for the involvement of tyrosine-69 in the control of stereospecificity of porcine pancreatic phospholipase A sub(2)

The authors have studied the role of Tyr-69 of porcine pancreatic phospholipase A sub(2) 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 k sub(cat) and K sub(m). The data suggest that in porcine pancreatic phospholipase A sub(2) 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.