Chemical Modification of Lys‐6 in Phospolipase A₂ from Naja melanoleuca Snake Venom

Abstract

Phospholipase A2 isolated from Naja melanoleuca snake venom was modified with 4-chloro-3,5-dinitrobenzoate. The modification reaction was accelerated by the cofactor Ca2+. Micellar concentrations of the substrate analog n-tetradecylphosphocholine retarded the inactivation of the enzyme. Modification resulted in the incorporation of one mole dinitrobenzoate/mole of protein. The modified residue could be identified as Lys-6 in the amino acid sequence of the enzyme. Although the modified enzyme still retains a high affinity for micellar substrate analogs and the cofactor Ca2+, it had lost nearly all its activity towards micellar substrates. Upon reduction of the nitro groups in the modified protein the enzymatic activity could be restored by more than 50%. Therefore Lys-6 cannot be directly involved in enzymatic catalysis. It is concluded that introduction of the negatively charged dinitrobenzoate group on Lys-6 gives rise to a distortion of the active site of the enzyme.

In VitroandIn VivoEvaluation of Polyherbal Formulation against Russell’s Viper and Cobra Venom and Screening of Bioactive Components by Docking Studies

“…In addition aristolochic acid is a competitive inhibitor of type I PLA 2 and showed noncompetitive inhibition in the case of type II PLA 2 from russell's viper venom [37, 38]. Type II PLA 2 venom enzyme differs from type I PLA 2 by having extended C-terminal tail [39]. This leads to differance in their pharmacological sites of the enzyme.…”

“…Lys6 residues are found in PLA 2 of all Elapidae venom. It is reported that Lys6 plays an indirect role in the Ca 2+ cofactor for catalyst activity of the PLA 2 in snake venom by means of intermolecular H-bond with Ca 2+ catalytic active site residues [39]. Modification in the Lys6 might distort the binding ability of PLA 2 for substrate and cause a drastic loss in enzymatic activity [40].…”