Interaction of Monodispersed and Micellar Phospholipids with an Agkistrodon halys blomhoffii Phospholipase A2, in which the α-Amino Group Had Been Modified to an α-Keto Group

The pH dependence of the bmding constant of Ca2+ to a phospholipase A2 of Agkistrodon halys blomhoffii, in which the α-amino group had been selectively modified to an α-keto group, was studied at 25°C and ionic strength 0.1 by the tryptophyl fluorescence method. The dependence was compared with the results for the intact enzyme (Iketh et al. (1981) J. Biochem. 90, 1125–1130). The pH-dependence curve could be well interpreted in terms of the participation of the two ionizable groups Asp 49 and His 48, with pK values of 4.70 and 6.69, respectively. These values were slightly different from the respective pK values for the intact enzyme, 5.15 and 6.45. Ca2+ binding to the intact enzyme involves the participation of an additional ionizable group with a pK value of 7.30, which was thus assigned as α-amino group. The pH dependence of the binding constant of monodispersed n-dodecylphos-phoryicholine (n-C12PC) to the α-NH2-modified enzyme was studied at 25°C and ionic strength 0.1 by the aromatic circular dichroism (CD) method. The pH- dependence curve for the modified apoenzyme was interpreted as reflecting the participation of a single ionizable group with a pK value of 4.7, which was assigned to Asp 49 (to which a Ca2+ ion can coordinate) since the curve for the Ca2+ complex lacked this transition: the binding constant was independent of pH. The pH- dependence curves for the intact apoenzyme and its Ca2+ complex involve the participation of an additional ionizable group with pK values of 7.30 and 6.30, respectively (Ikeda & Samejima (1981) J. Biochem. 90, 799–804), which was assigned as the a-ammo group. The hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC8PC), catalyzed by the intact and the α-NH2-modified enzymes was studied by the pH stat method at 25°C, pH 8.2, and ionic strength 0.1