Characterization of phospholipase A₂ (PLA₂) from Taiwan cobra: Isoenzymes and their site-directed mutants

Extracellular and secretory phospholipase A₂ (PLA₂), a class of phospholipid digesting enzyme, is widely distributed in animal venoms of reptiles and insects. Two cDNAs encoding PLA₂ isoenzymes from Taiwan Cobra (Naja naja atra) were cloned into pQE-30 plasmid vector and expressed in Escherichia coli. The recombinant products were subjected to refolding using sulfonation under reduction/oxidation conditions with glutathione and enterokinase removal of His-tag, resulting in the active recombinant PLA₂ with the same molecular masses of native enzymes as determined by mass spectrometry. The recombinant PLA₂ was also shown by circular dichroism to possess a secondary structure similar to native PLA₂. The enzymatic activity of the major isoenzyme (PLA_2-1) is higher than the other minor isoenzyme (PLA_2-2), which shows two amino acid difference from PLA_2-1. Site-directed mutagenesis was used to probe the structure/function relationship of two highly conserved residues among all reported PLA₂, i.e., His-47 and Asp-93. Replacement of His-47 residue by either Ala or Arg resulted in the complete loss of activity. Similarly, the mutant Asp-93→Asn (D93N) also retained little activity. These results suggest that both His-47 and Asp-93 are essential for the catalytic activity of PLA₂. Computer graphic study, based on homology modelling, highlights the differences between native PLA₂ isoenzymes and their site-directed mutants, which may account for the differences in the observed biological activity.