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

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The pH dependence of the binding constant of Ca ₂₊ 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 (Ikeda 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-dodecylphosphorylcholine (n-C₁₂PC) to the α-NH_2-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 α-amino group. The hydrolysis of monodispersed 1, 2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC₆PC), catalyzed by the intact and the α-NH_-2modified enzymes was studied by the pH stat method at 25°C, pH 8.2, and ionic strength 0.1 in the presence of 3 mM Ca²⁺. The Km value for the modified enzyme was found to be very similar to that for the intact enzyme: this was compatible with the results of the direct binding study on the monodispersed n-C₁₂PC under the same conditions. However, the kcat value was about 43% of the value for the intact enzyme, suggesting that the α-keto group introduced by the chemical modification perturbed the network of hydrogen bonds in the active site.
The pH dependence of the binding constant of micellar n-hexadecylphosphorylcholine (n-C₁₆PC) to the α-NH_2-modified enzyme was studied at 25°C and ionic strength 0.1 by the tryptophyl fluorescence method and was compared with the result for the intact enzyme (Ikeda et al. (1984) J. Biochem. 96, 1427-1436). At neutral and alkaline pH values, the binding constants for the modified apoenzyme and its Ca²⁺ complex were smaller than the corresponding constants for the intact enzyme (about 56% and 34%, respectively, at infinitely alkaline pH values). Nevertheless, the pH-dependence curves could be interpreted in terms of the participation of Asp 49 (and His 48) or His 48 in way similar to that for the intact enzyme, indicating no significant participation of the ionization of the α-amino group in the binding of micellar substrates: this contrasts with the result on the binding of the monodispersed substrates. In this connection, it was found that the enzymatic activity of the α-NH_2-modified enzyme toward egg-yolk suspensions was much smaller than that for the intact enzyme (only about 14.5%). The α-keto groups, introduced by the chemical modification, perturbed the network of hydrogen bonds in the active site and changed not only the kcat value but also the Km value.