Kinetics of the hydrolysis of mixed micelles of dipalmitoyllecithin with triton X-100 catalyzed by a phospholipase A2 from the venom of Agkistrodon halys blomhoffii.
The pH dependence of kinetic parameters for the hydrolysis of mixed micelles of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (diC16PC) with Triton X-100, catalyzed by the intact and the N-terminal alpha-NH2-modified phospholipases A2 (PLA2s) of Agkistrodon halys blomhoffii, was studied at 25 degrees C and ionic strength 0.1 in the presence of saturating amounts of Ca2+. The pH dependence of the kinetic parameters for the hydrolysis of monodispersed diC6PC, catalyzed by the modified enzyme, was also studied under the same conditions, and the data were compared with the previous results for the intact enzyme [Teshima, K. et al. (1986) J. Biochem. 100, 1655-1662]. The pK values of the catalytic group, His 48, and Tyr 52 were found to shift from 5.55 to 7.00 and from 10.50 to 11.50, respectively, on binding of the micellar substrates to the enzyme. On the other hand, no participation of these ionizable groups was observed for the binding of the monodispersed substrate. On the basis of the present finding and the X-ray crystallographic studies on bovine pancreatic PLA2 [Dijkstra, B.W. et. al. (1981) J. Mol. Biol. 147, 97-123] and on a PLA2 of Crotalus atrox venom [Brunie, S. et al. (1985) J. Biol. Chem. 260, 9742-9749], the hydrogen-bonding of Tyr 73, which is involved in the lipid-water interface recognition site, to His 48 and Tyr 52 in the active center was strongly suggested to be important for the hydrolysis of micellar substrates.
Teshima
K
K
Department of Hygienic Chemistry, Faculty of Pharmacy, Hoshi University, Tokyo.
Samejima
Y
Y
Kawauchi
S
S
Ikeda
K
K
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry*; D002384:Catalysis; D003435:Crotalid Venoms / Q000032:analysis*; D003461:Crystallography; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008823:Micelles; D017830:Octoxynol; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D011092:Polyethylene Glycols; D014961:X-Ray Diffraction
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D008823:Micelles; D015060:1,2-Dipalmitoylphosphatidylcholine; D011092:Polyethylene Glycols; D017830:Octoxynol; D010741:Phospholipases A; D054467:Phospholipases A2