pH Dependence of the Binding Constant of Ca2+ to Cobra Venom Phospholipases A21

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Abstract
The pH dependence of the binding constant of Ca2+ to phospholipases A2 [EC 3.1.1.4] of N. naja siamensis, N. naja kaouthia, and N. naja atra was studied at an ionic strength of 0.1 and 25°C by measuring the decrease in tryptophyl fluorescence. All three enzymes exhibited precisely the same pH dependence. The binding constants at various pH values of the enzyme of N. naja naja, reported by Roberts et al. ((1977) J. Biol. Chem. 252, 6011-6017), fell practically on our pH dependence curve. The pK value of an ionizable group was perturbed from 7.55 to 7.25 and protonation of another group with a pK value of 5.4 competed with the binding of the Ca2+ ion. The former group was assigned as a His residue corresponding to His 48 and the latter as Asp 49 in the active site on the basis of X-ray crystallographic results on the bovine pancreas enzyme (Dijkstra et al. (1978) J. Mol. Biol. 124, 53-60; Verheij et al. (1980) Biochemistry 19, 743-750). The pH dependence curve for porcine pancreas phospholipase A2, which was reported by Pieterson et al. ((1974) Biochemistry 13, 1439-1445) but is not yet well understood, was analyzed in a similar way on the basis of the pK value of His 48 estimated from the pH dependence of the tryptophyl fluorescence (van Dam-Mieras et al. (1976) Nobel Symp. 34, 177-197). The data were found to be well interpreted in terms of pK shifts of the α-amino group from 8.4 to 7.9 and of His 48 from 6.6 to 5.6 and in terms of protonation of Asp 49 with a pK value of 5.35, which competes with the Ca2+ ion binding. The pK shift of His 48 is in good agreement with those reported recently for equine and bovine enzymes (Verheij et al. (1980) Biochemistry 19, 743-750; Aguiar et al. (1979) Eur. J. Biochem. 100, 511-518). The pH dependences of fluorescence intensity at 350 nm (excited at 290 nm) of a cobra phospholipase A2 of N. naja siamensis and its complex with Ca2+ were studied at an ionic strength of 0.1 and 25°C. Two other cobra enzymes also showed similar pH dependences. The curves were well interpreted in terms of participations of groups with pK values less than 2, 3.9, 9.75, and 11.1, in addition to the contributions from Asp 49 and His 48 perturbed by the Ca2+ ion binding.