Kinetics of the Hydrolysis of Monodispersed Dihexanoyllecithin Catalyzed by the Phospholipase A2 from Agkistrodon halys blomhoffii Venom

The hydrolysis of 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC5PC), catalyzed by the phospholipase A1 from the venom of Agkistrodon halys blomhoffii, was studied at 25°C and the ionic strength of 0.1 in the presence of 3–33.3 mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical micelle concentration (cmc), were analyzed according to the Michaelis-Menten equation. The pH-dependence curve of the Km value exhibited only one transition below pH 8. The analytical results indicated that the pK value of 6.30 of an ionizable group changed to 6.54 on the binding of the monodispersed substrate. This ionizable group was assigned as the a-amino group on the basis of its pK value, which had been determined from the pH dependence of the binding constant of monodispersed n-dodecylphosphorylcholine (n-C12PC) (Ikeda and Samejima (1981) J. Biochem. 90, 799–804, and Haruki et al. (1986) J. Biochem. 99, 99–109). The pH-dependence curve of the kcat value exhibited two transitions, below pH 6.5 and above pH 9.5. The analytical results indicated the participation of two ionizable groups with pK values of 5.55 and 10.50. Deprotonation of the former and protonation of the latter group were found to be essential for the catalysis. The former ionizable group was assigned as His 48 in the active site on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Ikeda et al. (1981) J. Biochem. 90, 1125–1130). The latter group was tentatively assigned as the invariant Tyr 52, which is located in close proximity to the irmdazole ring of His 48 (Dijkstra et al. (1983) J. Mol. Biol. 168, 163–179). These findings were compatible with the results of our previous study on the cobra (Naja naja atra) venom phospholipase A2, although in the case of the cobra enzyme the additional minor participation of the α-amino group was observed.