Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes
Introduction
Phospholipase A2 (PLA2) enzymes hydrolyze glycerophospholipids at the sn-2 position of the glycerol backbone releasing lysophospholipids and fatty acids. They occur ubiquitously in nature as both intracellular and extracellular forms and hydrolyze various phospholipids. They are the most studied among all phospholipases because of their pivotal role in various biological activities. Mammalian PLA2 enzymes play important role in fertilization (Fry et al., 1992), cell proliferation (Arita et al., 1991), smooth muscle contraction (Nakajima et al., 1992, Sommers et al., 1992), and hypersensitization and chronic inflammatory diseases (Vadas and Pruzanski, 1986, Vadas et al., 1993). They are also important in cellular functions such as signal transduction via biosynthesis of prostaglandins and leukotrienes, and membrane homeostasis including the maintenance of the cellular phospholipid pools and membrane repair through deacylation/reacylation (Verheij et al., 1981, Jain and Berg, 1989, Dennis et al., 1991, Kudo et al., 1993, Dennis, 1994). However, mammalian enzymes are generally nontoxic and fail to induce potent pharmacological effects. On the other hand, snake venom PLA2 enzymes, in addition to their possible role in the digestion of the prey, exhibit a wide variety of pharmacological effects by interfering in normal physiological processes (Kini, 1997a) (Table 1). Some of the most toxic and potent pharmacologically active components of snake venoms are either PLA2 enzymes or their protein complexes. For example, all known presynaptic neurotoxins from snake venom are PLA2 enzymes per se or contain PLA2 as an integral part (Gubenšek et al., 1997, Bon, 1997). PLA2 myotoxins are more potent and fast-acting than their nonenzymatic counterparts (Fletcher et al., 1997). The ability to induce pharmacological effects with higher potencies underscores the importance of PLA2 enzymes in snake venom toxicity.
So far, the amino acid sequences of over 280 PLA2 enzymes have been determined (Danse et al., 1997, Tan et al., 2003). (A database of snake venom PLA2 enzymes is available at http://sdmc.lit.org.sg/Templar/DB/snaketoxin_PLA2/index.html). Despite the differences in their pharmacological properties, they share 40–99% identity in their amino acid sequences and hence significant similarity in their three dimensional folding (Scott, 1997). Thus the functional differences among PLA2 enzymes cannot be easily correlated to their structural differences. The structural similarity makes the structure–function relationships subtle, complicated and challenging. The shared common ability to hydrolyze at the sn-2 position of phospholipids and the lack correlation between enzymatic activity and lethal toxicity or pharmacological potency (Rosenberg, 1997a, Rosenberg, 1997b), make the mechanisms by which snake venom PLA2 enzymes induce a wide spectrum of pharmacological effects intriguing. A monograph (Kini, 1997b) on snake venom PLA2 enzymes deals with their structure, function and mechanism. This review provides an overview of the structure–function relationships and the mechanisms of snake venom PLA2 enzymes and their implications.
Section snippets
Purification
The quality of enzyme preparations is crucial for structure–function and mechanistic studies. Any contamination may contribute significantly to the results; such data could be invalid and may complicate the studies on structure-function and mechanism. The following two factors should be considered during the purification of PLA2 enzymes from snake venoms: (a) Snake venoms often contain a large number of PLA2 isoenzymes. For example, Naja naja, Vipera russelli, Trimeresurus flavoviridis,
Catalysis
PLA2 enzymes are unique hydrolytic enzymes in that they are highly soluble in water but hydrolyze water-insoluble substrate phospholipids. They hydrolyze phospholipids in monomeric, micellar or lipid bilayer phases. They exhibit a large and abrupt increase (up to 10,000-fold) in their catalytic activity when monomeric phospholipids aggregate to form micelles at their critical micellar concentration (Verger and DeHaas, 1973, Verger et al., 1973, Jain and Cordes, 1973a, Jain and Cordes, 1973b,
Pharmacological effects
Snake venom PLA2 enzymes exhibit a wide variety of pharmacological effects (Table 1). When studying a new enzyme, it is important to examine its pharmacological effects in intact animals, rather than in isolated tissues or in vitro methods. The in vitro studies sometimes show nonspecific effects that are due to the inherent phospholipolytic activity resulting in wrong conclusions. Occasionally, some of the pharmacological effects can be studied only in in vitro systems.
In most cases, snake
Target model and pharmacological specificity
Although a variety of pharmacological effects are induced by PLA2 enzymes, not all the effects are exhibited by all PLA2 enzymes. Each enzyme exhibits a specific effect. For example, β-bungarotoxin, a PLA2 containing toxin, induces a presynaptic effect on nicotinic acetylcholine transmission (Strong et al., 1976) but not glutamate and adrenergic transmission even at high concentrations (Abe et al., 1977). It does not show postsynaptic activity (Yang, 1978), myotoxicity (Fraenkel-Conrat, 1982-83
Enzymatic activity and pharmacological effects
Upon binding to the target protein, PLA2 enzymes can induce their pharmacological effects through mechanisms that are either dependent on or independent of their enzymatic activity. In the mechanisms that are dependent on enzymatic activity, either the hydrolysis of intact phospholipids or the released products such as lysophospholipids and fatty acids can cause the pharmacological effect (For details, see Kini and Evans, 1989a). The inherent enzymatic activity can cause membrane damage and
Pharmacological sites
PLA2 enzymes bind to target proteins through specific pharmacological sites (Fig. 1) (Kini and Evans, 1989a). The presence of pharmacological sites is supported by chemical modification studies, polyclonal and monoclonal antibodies and interaction of inhibitors (for reviews, see Yang, 1997, Stiles and Choumet, 1997, Gowda, 1997). Various chemical methods to modify specific amino acid residues have been used to identify these structural features. Despite systematic efforts, it has been difficult
Accelerated evolution of molecular surface
Recently by comparing the structures of 127 snake venom PLA2 enzymes, we showed that mutational hot spots occur on the surface of this protein molecule (Kini and Chan, 1999). Natural substitutions occur about 2.6–3.5 times greater in fully exposed residues than in the buried residues. As described above, the surface residues are important in molecular recognition of target proteins. Thus, natural substitutions in the surface residues would contribute directly towards modifying the molecular
Future prospects
Snake venom PLA2 enzymes are small enzymes which cause havoc by interfering in the normal physiological processes of the victim and inducing a variety of pharmacological effects. They provide a great challenge to protein chemists as subtle and complex puzzles in structure-function relationships. A better understanding will contribute to our knowledge of protein–protein interactions, protein targeting and protein engineering and to the development of better targeted delivery systems. Further
Acknowledgements
I thank my colleagues and mentors who ‘supported’ and nurtured at various levels several ideas presented in this chapter. This work is supported by Academic research grants from the National University of Singapore.
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