PROBING THE INTERACTION OF PHOSPHOLIPASE A2 AND PHOSPHOLIPIDS WITH RECOMBINANT DNA TECHNIQUES

Abstract

The primary structure of eighty phospholipases A> from different sources is known. These sequences reveal a high degree of homology, and aboutthirty percent ofall residuesis fully conserved. In addition about twentyfive percentof the aminoacids is substituted by amino acids with similar properties like charge and/or polarity and hydrogen-bonding capacity. The use of recombinant DNAtechniques has proovento be a powerful techniqueto study the role of amino acid side chains in porcine pancreatic phospholipase Ap. The application of these techniques has madeit possible to create a deletion mutant with enhanced activity and improved crystallisation properties. It was shown that Leu-31 is important for the orientation of the substrate molecule and is probably also importantfor the shielding of the active site from excess water. Tyr- 69 was shownto be involved in the orientation of the substrate molecule through an interaction with the phosphate group ofthe substrate. A numberof absolute conserved residues like Tyr-52 and Tyr-73 are important for folding and/or stability of the protein. Theresults of the studies described here support the mechanism of catalysis of phospholipase A2 that was proposed already ten years ago.