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Modification of the head-group selectivity of porcine pancreatic phospholipase A-2 by protein engineering

, : Modification of the head-group selectivity of porcine pancreatic phospholipase A-2 by protein engineering. Biochemistry. 32(45): 12203-12208

On the basis of the three-dimensional structures of phospholipid and porcine pancreatic phospholipase A-2 (pla-2), it was predicted that the removal of a negative charge in the hydrophilic region of the phospholipid binding site would influence the head-group selectivity of porcine pancreatic pla-2. To test this prediction, glutamic acid 46 was changed to leucine by site-directed mutagenesis. The E46L mutant, expressed in Escherichia coli, was purified and characterized. The mutation did not affect the activity toward the mixed micellar substrate, but the activity 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 carries no net charge in the head group. The native pla-2 was inhibited by the product(s) released from DiC-12-P but not the mutant enzyme. Kinetic analysis revealed that the E46L mutant and the native pla-2 had comparable affinities (K-m) toward monomeric and micellar phospholipids of zwitterionic type while the activity (k-cat) of E46L, toward the same substrates, was approximately 50% lower compared to that of native pla-2. 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 higher than those of native pla-2. However, the kinetic parameters of mutant and native pla-2s remained unchanged for monomeric HEPG, with one negative charge in the head group. Thus, we have modified the head-group selectivity of porcine pancreatic pla-2 by protein engineering.


PMID: 8105891

DOI: 10.1021/bi00096a033

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