STRUCTURAL ASPECTS OF INTERFACIAL ADSORPTION - A CRYSTALLOGRAPHIC ANDSITE-DIRECTED MUTAGENESIS STUDY OF THE PHOSPHOLIPASE A(2) FROM THE VENOM OF AGKISTRODON PISCIVORUS PISCIVORUS

Recent genetic and structural studies have shed considerable light on the mechanism by which secretory phospholipases A(2) interact with sub strate aggregates. Electrostatic forces play an essential role in opti mizing interfacial catalysis. Efficient and productive adsorption of t he Class I bovine pancreatic phospholipase A(2) to anionic interfaces is dependent upon the presence of two nonconserved lysine residues at sequence positions 56 and 116, implying that critical components of th e adsorption surface differ among enzyme species (Dua, R. Wu, S.-K., a nd Cho, W. (1995) J. Biol. Chem. 270, 263-268). In an effort to furthe r characterize the protein residues involved in interfacial catalysis, we have determined the high resolution (1.7 Angstrom) x-ray structure of the Class II Asp-49 phospholipase A(2) from the venom of Agkistrod on piscivorus piscivorus, Correlation of the three-dimensional coordin ates with kinetic data derived from site-directed mutations near the a mino terminus (E6R, K7E, K1OE, K11E, and K16E) and the active site (K5 4E and K69Y) defines much of the interface topography, Lysine residues at sequence positions 7 and 10 mediate the adsorption of A. p. pisciv orus phospholipase A(2) to anionic interfaces but play Little role in the enzyme's interaction with electrically neutral surfaces or in subs trate binding. Compared to the native enzyme, the mutant proteins K7E and K1OE demonstrate comparable (20-fold) decreases in affinity and ca talysis on polymerized mixed liposomes of l-2-(1-pyrenedecanoyl)-sn-gl ycero-3-phosphocholine and ipoyloxy)dodecanoyl]-sn-glycero-3-phosphogl ycerol, while the double mutant, K7E/K1OE, shows a more dramatic 500-f old decrease in catalysis and interfacial adsorption, The calculated c ontributions of Lys-7 and Lys-10 to the free energy of binding of A. p . piscivorus phospholipase A(2) to anionic liposomes (-1.8 kcal/mol at 25 degrees C per lysine) are additive (i.e. -3.7 kcal/mol) and togeth er represent nearly half of the total binding energy, Although both ly sine side chains lie exposed at the edge of the proposed interfacial a dsorption surface, they are geographically remote from the correspendi ng interfacial determinants for the bovine enzyme. Our results confirm that interfacial adsorption is largely driven by electrostatic forces and demonstrate that the arrangement of the critical charges (e.g. ly sines) is species-specific. This variability in the topography of the adsorption surface suggests a corresponding flexibility in the orienta tion of the active enzyme at the substrate interface.