MODIFICATION OF THE HEADGROUP SELECTIVITY OF PORCINE PANCREATIC PHOSPHOLIPASE-A(2) BY PROTEIN ENGINEERING

On the basis of the three-dimensional structures of phospholipid and p orcine pancreatic phospholipase A2(pla2), it was predicted that the re moval of a negative charge in the hydrophilic region of the phospholip id binding site would influence the head-group selectivity of porcine pancreatic pla2. To test this prediction, glutamic acid 46 was changed to leucine by site-directed mutagenesis. The E46L mutant, expressed i n Escherichia coli, was purified and characterized. The mutation did n ot affect the activity toward the mixed micellar substrate, but the ac tivity of E46L toward DiC-12-P, which has two negative charges on the head group, was three times higher than that of DiC-12-PC, which carri es no net charge in the head group. The native pla2 was inhibited by t he product(s) released from DiC-12-P but not the mutant enzyme. Kineti c analysis revealed that the E46L mutant and the native pla2 had compa rable affinities (K(m)) toward monomeric and micellar phospholipids of zwitterionic type while the activity (k(cat)) of E46L, toward the sam e substrates, was approximately 50% lower compared to that of native p la2. When micellar DiC-12-P was used as a substrate, the K(m)app value for E46L was four times lower and the k(cat)app/K(m)app was 5-fold hi gher than those of native pla2. However, the kinetic parameters of mut ant and native pla2s remained unchanged for monomeric HEPG, with one n egative charge in the head group. Thus, we have modified the head-grou p selectivity of porcine pancreatic pla2 by protein engineering.