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 1H 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Dupureur
C M
CM
Department of Chemistry, Ohio State University, Columbus 43210.
Yu
B Z
BZ
Mamone
J A
JA
Jain
M K
MK
Tsai
M D
MD
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000032:analysis; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism; D054467:Phospholipases A2; D011487:Protein Conformation; D015202:Protein Engineering; D013329:Structure-Activity Relationship
D010649:Phenylalanine; D010741:Phospholipases A; D054467:Phospholipases A2