Snake venom PLA₂ enzymes are esterolytic enzymes which hydrolyze glycerophospholipids at the sn-2 position of the glycerol backbone releasing lysophospholipids and fatty acids. So far, several hundred snake venom enzymes have been purified and characterized. They share similarity in structure and catalytic function with mammalian enzymes. However, in contrast to mammalian enzymes, many are toxic and induce a wide spectrum of pharmacological effects. Therefore their structure-function relationships are subtle and complicated. We developed theoretical methods to solve structure-function relationships of snake venom PLA₂ enzymes. The following are some of our contributions to the structure-function relationships of PLA₂ enzymes.

• Presynaptic neurotoxic site – Several groups predicted the neurotoxic site of PLA₂ enzymes by theoretical methods. Dufton and his coworkers were the first to predict the site responsible for b -neurotoxicity by direct homology studies between neurotoxic and non-neurotoxic PLA₂ enzymes. Based on the hydropathic profiles of PLA₂ enzymes, we predicted hydrophobic helix E may be important for the presynaptic neurotoxicity based on 26 amino acid sequences. Recent analysis of hydropathic profiles of 40 single chain neurotoxic PLA₂ enzymes showed hydrophobic segment in the region of 80-110 residues. However, several of the 47 non-neurotoxic PLA₂ enzymes also showed similar hydrophobicity indicating the lack correlation between the neurotoxicity and hydrophobicity.

• Myotoxic region – By charge density distribution, we identified a myotoxic site in presynaptically active PLA₂ enzymes. There is a characteristic cationic site, +00+++00+, in myotoxic enzymes whereas nonmyotoxic enzymes do not contain this site. This cationic site is located towards the amino terminal side of the hydrophobic helix E, and both these sites together form the complete myotoxic region. A similar combination of hydrophobic and cationic sites was also found in nonenzymatic myotoxins. Subsequently, a hydrophobic segment flanking the cationic site is commonly found in various classes of cytolysins, including myotoxins, hemolysins and antibacterial peptides (Common cytolytic region ).

• Anticoagulant region – By a systematic and direct comparison of the amino acid sequences of strong, weak and non-anticoagulant enzymes, we identified the  anticoagulant region between residues 54 and 77. This region is positively charged in strongly anticoagulant PLA₂ enzymes. At both ends of the region is a pair of Lys residues that are replaced by neutral or negatively charged amino acid residues. All the chemical modification studies supported this prediction. The predicted anticoagulant site is strongly supported by site-directed mutagenesis as well as using synthetic peptides.

 

 

 

Key Publications