title: Kinetics of the hydrolysis of monodispersed dihexanoyllecithin catalyzed by a cobra (Naja naja atra) venom phospholipase A2.
abstract: 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 degrees C ionic strength 0.1 in the presence of 3-10 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 micellar concentration (cmc), were analyzed according to the Michaelis-Menten equation. The Km 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. The pH dependence curve of the kinetic parameter, kcat/Km, 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).(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D001665:Binding Sites; D004546:Elapid Venoms / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010713:Phosphatidylcholines / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000494:pharmacology; D011487:Protein Conformation; D014961:X-Ray Diffraction
chemical_list: D004546:Elapid Venoms; D010713:Phosphatidylcholines; D010767:Phosphorylcholine; C032166:1,2-hexanoylphosphatidylcholine; C028810:dodecylphosphocholine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2