Kinetics of the hydrolysis of monodispersed dihexanoyllecithin catalyzed by a cobra (Naja naja atra) venom phospholipase A2.

The hydrolysis of 1, 2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC₆PC), catalyzed by a cobra (Naja naja atra) venom phospholipase A₂, was studied at 25°C and ionic strength 0.1 in the presence of 3-10mM 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 micellar concentration (cmc), were analyzed according to the Michaelis-Menten equation. The K_m value was practically independent of pH (between pH 6.75 and 10.30). This finding was consistent with the result of a direct binding study on monodispersed n-alkylphosphorylcholines (Teshima et al. (1981) J. Biochem. 89, 1163-1174). The hydrolysis of the substrate was competitively inhibited by the presence of monodispersed n-dodecylphosphorylcholine (n-C₁₂PC). These results indicated that the substrate and n-C₁₂PC compete for the same site on the enzyme molecule.<br> The pH dependence curve of the kinetic parameter, k_cat/K_m, exhibited three transitions, below pH 8, between pH 8 and 9.5, and above pH 10. The analysis indicated the participation of three ionizable groups with pK values of 7.25, 8.50, and 10.4. The deprotonation of the first group and the protonation of the third group were found to be essential for the catalysis. The first 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⁺ (Teshima et al. (1981) J. Biochem. 89, 13-20). The second 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 rate constant for the reaction of p-bromophenacyl bromide (BPB) with His 48 (Teshima et al. (1984) J. Biochem. 96, 1903-1913). The third group was tentatively assigned as the invariant Tyr 52, located in close proximity to the imidazole ring of His 48.