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PDBsum entry 2bpp
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Carboxylic ester hydrolase
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PDB id
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2bpp
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References listed in PDB file
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Key reference
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Title
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Phospholipase a2 engineering. X-Ray structural and functional evidence for the interaction of lysine-56 with substrates.
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Authors
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J.P.Noel,
C.A.Bingman,
T.L.Deng,
C.M.Dupureur,
K.J.Hamilton,
R.T.Jiang,
J.G.Kwak,
C.Sekharudu,
M.Sundaralingam,
M.D.Tsai.
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Ref.
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Biochemistry, 1991,
30,
11801-11811.
[DOI no: ]
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PubMed id
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Abstract
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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., Zürcher-Neely, H., Reardon, I.M.,
Oriaku, E., Kézdy, 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.
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