A model structure of Naja naja kaouthia cobra venom phospholipase A2 has been constructed by utilizing molecular modeling techniques. Analysis of the model and available biochemical data reveal the presence in this enzyme of a putative recognition site for choline derivatives in loop 57-70 made up of residues Trp-61, Tyr-63, Phe-64, and Lys-65, together with Glu-55. The magnitude and shape of the electrostatic potential in this binding site are approximately 80% similar to that in the McPC603 antibody binding site specifically recognizing phosphocholine. Docking studies indicate that the recognition site we now describe and the phosphocholine head of an n-alkylphosphocholine molecule are complementary both sterically and electronically, mainly due to anion-cation and cation-pi interactions. Moreover, binding enthalpies of n-heptylphosphocholine to this site are found to parallel the catalytic rate of pancreatic, mutant pancreatic, and cobra venom phospholipase A2 enzymes acting on dihexanoylphosphatidylcholine micelles, suggesting that it behaves as an activator site. This proposal is in keeping with the "dual phospholipid" model put forward to account for the phenomenon of interfacial activation. This novel site is also shown to be able to discriminate choline derivatives from ethanolamine derivatives, in accord with experimental data. On the basis of the results obtained, two functions are assigned to this putative activator site: (i) desolvation of the lipid-enzyme interface, particularly the surroundings of tyrosine at position 69 (Tyr-63), and (ii) opening of the entrance to the active site by means of a conformational change of Tyr-63 whose chi 2 angle rotates nearly 60 degrees.
A molecular model of the interaction between manoalide (MLD) and bee venom phospholipase A2 (bv-PLA2) has been derived making use of a combination of computational methods. MLD was built in its open form and simulated by using molecular dynamics techniques. It is shown that the polar part of the molecule, which is thought to be the reactive region, is endowed with considerable conformational flexibility whereas the apolar region is rather rigid. The proposed active conformation of MLD and the main putative binding site for MLD on this enzyme were identified by matching potential energy GRID maps for both ligand and receptor with the chemical structure of the respective counterpart. The binding site is found in the C-terminal region of bv-PLA2, forming part of the proposed interfacial surface for binding to aggregated substrates, and comprises two distinct regions: (i) a hydrophobic cavity delimited by the C-terminal beta-sheet and the antiparallel beta-sheet, which interacts with the apolar zone of MLD, and (ii) a cationic site made up of residues Arg-58 and Lys-94, which interacts with the polar zone. Molecular dynamics and molecular orbital calculations indicate that the most likely initial reaction between MLD and bv-PLA2 is formation of a Schiff base between Lys-94 and the aldehyde generated upon opening of MLD's gamma-lactone ring, supporting recent model reaction studies. The inhibition seems to be a consequence of the occupation by MLD of a site overlapping a phosphocholine binding site in bv-PLA2 presumably involved in the interface desolvation process. The present model represents a starting point for further structural studies on the mechanism of phospholipases A2 inactivation by MLD and MLD-like compounds.
Mammalian nonpancreatic secretory phospholipase A2 (PLA2) splits the 2-acyl bond in 1,2-diacylphosphatides.* 1 This enzyme has been found in high concentrations in the synovial fluid of patients with rheumatoid arthritis,2 and it has been suggested that inhibitors of this enzyme may have therapeutic value. The three-dimensional structure of human synovial fluid PLA2 (HSF-PLA2) is known both
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