A pH-Induced Dissociation of the Dimeric Form of a Lysine 49-Phospholipase A2Abolishes Ca2+-Independent Membrane Damaging Activity

Abstract

The hydrolysis of phospholipids by class II phospholipase A₂ (PLA₂) involves a Ca²⁺ ion cofactor bound to the Asp49 residue in the active site region. In the lysine 49 phospholipase A₂ homologues (Lys49-PLA₂), the Asp49 residue is substituted by Lys, and consequently the Lys49-PLA₂s show no Ca²⁺ binding and no detectable phospholipid hydrolysis. Nevertheless, the Lys49-PLA₂s demonstrate membrane damaging activity by an incompletely understood Ca²⁺-independent mechanism of action. Using a combination of steady-state and time-resolved fluorescence techniques, we have examined the effect of pH on the monomer−dimer equilibrium of bothropstoxin I (BthTX-I), a Lys49-PLA₂ from the venom of Bothrops jararacussu which contains a single Trp77 residue located at the dimer interface. At pH 5.0, we observe a decreased quantum yield, a decreased rotational correlation time, and an increased bimolecular quenching rate constant with iodide. These results are consistent with a pH-induced dissociation of the BthTX-I dimer, with the consequent exposure of the Trp77 residue to aqueous solvent. In the presence of liposomes, membrane damaging activity is observed only under conditions in which the dimeric form of the BthTX-I is favored. These results demonstrate that the dimeric form of the protein is essential for the initiation of the Ca²⁺-independent membrane damaging activity.