Identification of the Tryptophan Residue Located in the Calcium Binding Site of Trimeresurus flavoviridis Phospholipase A2

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Abstract
When phospholipase A2 from the venom of Trimeresurus flavoviridis (the Habu snake) was oxidized with N-bromosuccinimide at pH 4.0, its activity decreased linearly with increase in the extent of oxidation of tryptophan residues. Oxidation of two of the four tryptophan residues caused an apparent loss of activity. The accessibilities of the tryptophan residues were analyzed with differently oxidized phospholipase A2 preparations and were determined to be in the following order: Trp-3∼Trp-30>Trp-68>Trp-108. The magnitude of the difference spectrum with a negative peak at 292 nm which is produced upon the binding of Ca2+ in the vicinity of tryptophan residues) decreased in a concave manner with increase in the extent of oxidation of tryptophan residues and was greatly diminished when 2 mol of tryptophan residues were oxidized. The activity and Ca2+-induced difference spectrum are thus related to either Trp-3 or Trp-30 or both. Des-octapeptide(1–8)-phospholipase A2 (L-fragment) is 14% as active as phospholipase A2 and is able to give a Ca2+-induced difference spectrum which is smaller than, but similar to, that of phospholipase A2. Its activity and the magnitude of the Ca2+-induced difference spectrum decreased along similar paths with increase in the amount of tryptophan residues oxidized, but in a manner indicating that two tryptophan residues are apparently responsible for the activity and the Ca2+-induced difference spectrum. The order of accessibility of the tryptophan residues of L-fragment was Trp-30∼Trp-108>Trp-68. Trp-108, however, could be excluded from the residues located in the active site by reference to the tertiary structure of homologous Crotalus atrox phospholipase A,. Thus, Trp-30 is located in the Ca2+ binding site and is responsible for the activity of L-fragment. It is thus concluded that in phospholipase A2 Trp-30 is located in the Ca2+ binding site. From the concave decrease of relative magnitude of the Ca2+-induced difference spectrum and the linear decrease of relative activity upon oxidation of phospholipase A2, it may be assumed that both Trp-3 and Trp-30 are required to produce the Ca2+-induced difference spectrum, while only Trp-30 need be intact for activity. Anomalous binding of Ca2+ was observed for oxidized phospholipase A2.