Kinetics of the hydrolysis of monodispersed dihexanoylphosphatidylcholine catalyzed by bovine pancreatic phospholipase A2: roles of Ca2+ binding and ionizations of amino acid residues in the active site.
Phospholipases A2 (PLA2s) are classified into two groups, I and II, according to differences in the polypeptide-chain length and intramolecular disulfide bondings. The hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by bovine pancreas PLA2 (Group I) was studied at 25 degrees C and ionic strength 0.2, and the initial velocity data were analyzed by means of the Michaelis-Menten equation. The Michaelis constant, Km, was found to be practically independent of Ca2+ concentration and also of pH value. The latter result indicates no participation of the ionizable groups in the active site in the substrate binding. The pH-dependence curve of the logarithm of the catalytic center activity, kcat, obtained in the presence of a practically saturating amount of Ca2+, showed three transitions ascribable to the participation of three ionizable groups with pK values of 5.00, 8.40, and 9.50. The respective groups were tentatively assigned to the catalytic group His 48, the N-terminal alpha-amino group, and invariant Tyr 52, which is located in close proximity to the imidazole ring of His 48. Deprotonation of His 48 and protonation of Tyr 52 were shown to be essential to the catalysis. The importance of the ionization state of the alpha-amino group was also indicated.(ABSTRACT TRUNCATED AT 250 WORDS)
Fujii
S
S
Department of Biochemistry, Osaka University of Pharmaceutical Sciences.
Inoue
T
T
Inoue
S
S
Ikeda
K
K
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D066298:In Vitro Techniques; D007477:Ions; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D010713:Phosphatidylcholines / Q000378:metabolism*; D010741:Phospholipases A / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D054467:Phospholipases A2
D000596:Amino Acids; D007477:Ions; D010713:Phosphatidylcholines; C032166:1,2-hexanoylphosphatidylcholine; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium