Mechanistic insights into NOBA hydrolysis by viper venom secreted phospholipase A2

Snake envenoming remains a major global health challenge, particularly in tropical and subtropical regions. Among the key toxic components of snake venom, secreted phospholipases A₂ (sPLA₂) play a crucial role by hydrolysing cell membrane phospholipids, leading to membrane disruption and severe toxic effects such as inflammation, neurotoxicity, and myotoxicity. To study sPLA₂ catalytic activity, synthetic soluble substrates like 4-nitro-3-octanoyloxy benzoic acid (NOBA) are widely used in experimental assays, in alternative to membrane phospholipids. However, it is questionable whether mechanistic conclusions taken with small, soluble substrates can be extrapolated to true cell-membrane substrates. Here, we employed QM/MM calculations to investigate the catalytic mechanism of sPLA₂ using NOBA as a substrate. Our focus was on a sPLA₂ from Bothrops asper. However, the high conservation of sPLA₂ active sites suggests our conclusions should be generalisable to the sPLA₂ of most snake species. The results reveal a mixed single-water/assisted water mechanism. First, a water molecule, deprotonated by His₄₇, performs a nucleophilic attack on NOBA's carbonyl carbon, with a free energy barrier of 12.8 kcal/mol, resembling the single-water pathway. The collapse of the tetrahedral intermediate and protonation of the leaving group involves two water molecules, resembling the assisted-water pathway. This mixed pathway highlights the catalytic versatility of sPLA₂ and offers new insights into its enzymatic activity with synthetic substrates. Importantly, our finding support NOBA as a valid and suitable substrate for studying the chemical component of sPLA₂ toxicity, even though it does not account for the equally important membrane-binding component of the toxicity mechanism.