PHOSPHOLIPASE-A2 ENGINEERING - THE STRUCTURAL AND FUNCTIONAL ROLES OF AROMATICITY AND HYDROPHOBICITY IN THE CONSERVED PHENYLALANINE-22 AND PHENYLALANINE-106 AROMATIC SANDWICH

The highly conserved phenylalanine-22 and phenylalanine-106, arranged as an aromatic sandwich, form part of an invariant hydrophobic wall that shields the active site of bovine pancreatic phospholipase A2 (PLA2) from bulk solvent [Dijkstra, B. W., Drenth, J., & Kalk, K. H. (1981) Nature 289, 604-606]. The residues have also been suggested to interact with the sn-2 acyl chain of bound phospholipid substrate [White, S. P., Scott, D. L., Otwinowski, Z., Gelb, M. H., & Sigler, P. B. (1990) Science 250,1560-1563]. We now report the importance of these two residues in the structure and function of PLA2 in terms of aromaticity (changing to Ile) and hydrophobic (changing to Ala) and hydrophilic (changing to Tyr) character of these residues. The structural properties of the mutants were analyzed by proton NMR and by guanidine hydrochloride-induced denaturation. The functional properties were determined by measuring kinetic parameters toward various substrates in the forms of monomers, micelles, and vesicles, and by measuring equilibrium dissociation constants at the interface. The results show that (i) The conformational stability of each mutant was as good as that of wild-type PLA2; none of the mutants was significantly perturbed structurally as judged from detailed H-1 NMR analysis. These results suggest that neither the Phe-22/Phe-106 face-to-face pair nor the Phe-22/Tyr-111 edge-to-face pair plays a significant structural role. (ii) Mutations to Ile at either position 22 or position 106 resulted in only minor perturbations in activity. This suggests that the aromaticity is not important to the function of these two residues. (iii) Substitution at either of these two positions with Ala resulted in 10-200-fold decreases in catalytic activities, suggesting that the hydrophobic bulk of both residues is important for catalysis, possibly by interacting with the sn-2 acyl chain. (iv) Replacement of Phe-106 by Tyr resulted in decreased activity; the same substitution at position 22 produced an enzyme with wild-type activity. The differential behavior of the two mutants can be rationalized by the orientation of the side chains: Phe-22 points outward facing solvent, whereas Phe-106 points into the active site. (v) Detailed analysis of all the mutants using scooting mode kinetics and equilibrium measurements suggested that the perturbation in the function of F22A, F106A, and F106Y lies primarily in k(cat); binding of the enzyme to the interface and binding of ligands to the enzyme at the interface are less perturbed.