2bpp
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PHOSPHOLIPASE A2 ENGINEERING. X-RAY STRUCTURAL AND FUNCTIONAL EVIDENCE FOR THE INTERACTION OF LYSINE-56 WITH SUBSTRATES
OverviewOverview
Site-directed mutagenesis studies of bovine pancreatic phospholipase A2, (PLA2, overproduced in Escherichia coli) showed that replacement of, surface residue Lys-56 by a neutral or hydrophobic amino acid residue, resulted in an unexpected and significant change in the function of the, enzyme. The kcat for phosphatidylcholine micelles increases 3-4-fold for, K56M, K56I, and K56F and ca. 2-fold for K56N and K56T but does not change, for K56R. These results suggest that the side chain of residue 56 has, significant influence on the activity of PLA2. In order to probe the, structural basis for the enhanced activity, the crystal structures of, wild-type and K56M PLA2 were determined by X-ray crystallography to a, resolution of 1.8 A. The results suggest that the mutation has not only, perturbed the conformation of the side chain of Met-56 locally but also, caused conformational changes in the neighboring loop (residues 60-70), resulting in the formation of a hydrophobic pocket by residues Met-56, Tyr-52, and Tyr-69. Docking of a phosphatidylcholine inhibitor analogue, into the active site of K56M, according to the structure of the complex of, cobra venom PLA2-phosphatidylethanolamine inhibitor analogue [White, S.P., Scott, D. L., Otwinowski, Z., Gleb, M. H., & Sigler, P. (1990) Science, 250, 1560-1563], showed that the choline moiety [N(CH3)3]+ is readily, accommodated into the newly formed hydrophobic pocket with a high degree, of surface complementarity. This suggests a possible interaction between, residue 56 and the head group of the phospholipid, explaining the enhanced, activities observed when the positively charged Lys-56 is substituted by, apolar residues, viz., K56M, K56I, and K56F. Further support for this, interpretation comes from the 5-fold enhancement in kcat for the mutant, K56E with a negatively charged side chain, where there would be an, attractive electrostatic interaction between the side chain of Glu-56 and, the positively charged choline moiety. Our results also refute a recent, report [Tomasselli, A. G., Hui, J., Fisher, J., Zurcher-Neely, H., Reardon, I.M., Oriaku, E., Kezdy, F.J., & Heinrikson, R.L. (1989) J. Biol., Chem. 264, 10041-10047] that substrate-level acylation of Lys-56 is an, obligatory step in the catalysis by PLA2.
About this StructureAbout this Structure
2BPP is a Single protein structure of sequence from Bos taurus with CA as ligand. Active as Phospholipase A(2), with EC number 3.1.1.4 Full crystallographic information is available from OCA.
ReferenceReference
Phospholipase A2 engineering. X-ray structural and functional evidence for the interaction of lysine-56 with substrates., Noel JP, Bingman CA, Deng TL, Dupureur CM, Hamilton KJ, Jiang RT, Kwak JG, Sekharudu C, Sundaralingam M, Tsai MD, Biochemistry. 1991 Dec 24;30(51):11801-11. PMID:1751497
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