Hydrolysis of micellar diheptanoylphosphatidylcholine catalyzed by bovine pancreatic phospholipase A2: kinetic characterization of group I and II enzymes.
Initial velocity data for the hydrolysis of micellar 1,2-diheptanoyl-sn-glycero-3-phosphorylcholine (diC7PC) catalyzed by bovine pancreatic PLA2 (Group I) were analyzed using the Michaelis-Menten equation. The Km value for the micellar substrate was found to be independent of Ca2+ concentration, as was the Km value for the monodispersed substrate. The pH dependence curve of Km in the presence of saturating amounts of Ca2+ showed two transitions reflecting large pK shifts of two ionizable groups from 5.0 to 5.45 and from 9.5 to 10.25, whereas the Km value for the monodispersed substrate was independent of pH [Fujii et al. (1991) J. Biochem. 110, 1008-1015]. The pH dependence curve of kcat showed three transitions, indicating the participation of three ionizable groups with pK values of 5.45, 8.4, and 10.25. Deprotonation of the first group and protonation of the third group were found to be essential for catalysis. The respective groups were assigned as the catalytic group His 48, the N-terminal alpha-amino group, and invariant Tyr 52. The present results as well as those for another Group I PLA2 (Naja naja atra) are very different from those for Group II PLA2s (Agkistrodon halys blomhoffii and Trimeresurus flavoviridis), which showed Ca(2+)-dependent substrate binding and no participation of the alpha-amino group in catalysis [Teshima et al. (1989) J. Biochem. 106, 518-527; Nishimura et al. (1992) J. Biochem. 111, 210-218].(ABSTRACT TRUNCATED AT 250 WORDS)
Hada
S
S
Department of Biochemistry, Osaka University of Pharmaceutical Sciences.
Fujii
S
S
Inoue
S
S
Ikeda
K
K
Teshima
K
K
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008823:Micelles; D010179:Pancreas / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2
D008823:Micelles; D010713:Phosphatidylcholines; C038470:1,2-heptanoylphosphatidylcholine; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium