Introduction of a C-terminal aromatic sequence from snake venom phospholipases A(2) into the porcine pancreatic isozyme dramatically changes the interfacial kinetics

Porcine pancreatic phospholipase A(2) (PLA(2)) was modified by single and m ultiple site-directed mutations at sites thought to be involved in interfac ial binding. Charged and polar residues in the C-terminal region were repla ced by aromatic residues on the basis of an analogy with snake venom PLA(2) s, which display high affinity for a zwitterionic interface. The PLA(2) var iants constructed were N117W, N117W/D119Y and K116Y/N117W/D119Y. Titration with micelles of a zwitterionic substrate suggests that the variants N117W and K116Y/N117W/D119Y possess improved ability to bind to the micellar subs trate interface, relative to the wild-type enzyme. Improved interfacial bin ding was confirmed by direct binding studies with micelles of a zwitterioni c substrate analogue, indicating up to five times higher affinity for both variants. Interfacial binding is not improved for the variant N117W/D119Y. Maximal enzyme velocities (V-max(app.)) with the zwitterionic substrate wer e between 25 and 75% of that of the wild-type enzyme. However, competitive inhibition and direct binding studies with a strong inhibitor revealed that the affinity for substrate present at the interface (K-m*) is perturbed by the mutations made. For the variant N117W, the slight decrease observed in V-max(app.) most likely made up of a 24-fold reduction in catalytic turnov er (k(cat)) and 18-fold improved substrate binding (K-m*).