Phospholipases A₂.

Phospholipases A₂ (PLA₂ - EC 3.1.1.4) are enzymes that specifically catalyze the hydrolysis the sn-2 acyl bonds of sn-3 phospholipids to release lysophospholipids and fatty acids [1] . The products of phospholipid hydrolysis may themselves be bioactive, or may serve as precursors for the synthesis of other bioactive compounds [2, 3] . Due to their central role in many cellular processes, PLA₂s from a variety of sources have been extensively studied, not only to understand the molecular bases of the catalytic mechanism and membrane binding [4] , but also with a view to understanding their regulatory functions within the cell [5, 6] . As a consequence of advances in molecular biology techniques, the number of recognized PLA₂s has increased dramatically in recent years, and these enzymes are currently classified into 12 groups (numbered I to XII) on the basis of disulphide bonding patterns and amino acid sequence similarity [7, 8] . The secreted group I/II PLA₂s are small (~14kDa), stable enzymes that are encountered in a wide variety of biological fluids and cells. Snake venoms are abundant sources of group I/II PLA₂, and although these venom derived PLA₂s (vPLA₂s) show a high level degree of structural conservation with mammalian secreted PLA₂s, they present remarkable diversity in terms of biological activities, and a given vPLA₂ may demonstrate multiple biological effects.

The hydrolysis of phospholipids by Group I/II PLA₂s involves a His48/Asp99 pair in the catalytic site together with a Ca²⁺ ion co-factor which is bound by Asp49 and main chain oxygens of the neighbouring calcium binding loop [9, 10] . It has been established that catalytic activity plays a key role in certain pharmacological effects of vPLA₂s [11, 12] , however it is now well proven that many pharmacological effects are at least partially independent of their hydrolytic activity [13-16] . The lack of a clear correlation between catalysis and pharmacological activity together with the diversity of biological effects raises the question as to the structural bases of these biological functions. Evidence is accumulating that suggests that these activities may be mediated by interactions between vPLA₂s and acceptors for endogenous PLA₂s on the membranes of the target cell [17-19] . The identification of additional mammalian PLA₂s [7] and the discovery of their protein acceptors in human cells [20-22] has expanded the number of potential targets and possible mechanisms of action for vPLA₂s.

Lysine 49-Phospholipases A₂.

A sub-family of Class IIA PLA₂s have been purified from several Asiatic and New World Viperid snake venoms in which the Asp49 residue is substituted by Lys [23, 24] . These Lys49-PLA₂s have been identified as abundant components of the venoms from New World Bothrops and Agkistrodon snake species [24] , in the Asiatic Trimereusrus species [25] and have been discovered more recently in additional New World viperid species, although in lesser quantities [26] . Not only is the distribution of the Lys49-PLA₂s more widespread than previously thought, but also the range of known biological effects is broader. In addition to myotoxic [24] , cytotoxic [27] and edema inducing [28, 29] effects, the Lys49-PLA₂s show pre-synaptic neurotoxicity [30] , stimulate the degranuation of mast cells [29] and directly influence leukocyte mobility [31] .

Initial reports suggested that the Lys49-PLA₂s retained low levels of catalytic activity [30, 32-34] , however subsequent studies with both native [23, 35, 36] and recombinant [37] proteins have failed to detect hydrolysis of phospholipid substrates. The crystal structures of Lys49-PLA₂s have demonstrated that the proteins have failed to detect hydrolysis of phospholipid substrates. The crystal structures of Lys49-PLA₂s have demonstrated that the e-amino group of the Lys49 is located in the position occupied by the Ca²⁺ co-factor in Asp49-PLA₂s [36, 38, 39] . It has been suggested that the reduction or elimination of catalytic activity results from either the re-orientation of the Cys29-Gly30 peptide bond in the calcium binding loop [38] , or reduced binding affinity of the Ca²⁺ co-factor [36] . Despite the absence of detectable catalytic activity, the Lys49-PLA₂s demonstrate a membrane damaging activity via a Ca²⁺-independent, non-hydrolytic mechanism [40, 41] . Bothropstoxin-I (BthTx-I) is a Lys49-PLA₂ isolated from the venom of Bothrops jararacussu which forms homodimers both in solution and in the crystalline state [42] . Using the BthTx-I as a model system, we have been using site directed mutagenesis to study the non-hydrolytic mechanism by which the Lys49-PLA₂s damage membranes, and the relevance of this mechanism in the myotoxic and bacteriocidal activities. 

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