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 (PLA2-1) is higher than the other minor isoenzyme (PLA2-2), which shows two amino acid difference from PLA2-1. 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.
Pan
F M
FM
Institute of Biochemical Sciences, National Taiwan University, Academia Sinica, Taipei, Taiwan 10098.
Chao
S C
SC
Wu
S H
SH
Chang
W C
WC
Chiou
S H
SH
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D017931:DNA Primers; D004546:Elapid Venoms / Q000201:enzymology*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004546:Elapid Venoms; D007527:Isoenzymes; D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2