REGULATION OF THE INTERACTION OF PANCREATIC PHOSPHOLIPASE A-2 WITH LIPID-WATER INTERFACES BY CA2+ IONS - MONOLAYER STUDY

In addition to the Ca2+ ion bound to the active site of porcine pancreatic phospholipase A2, it is known that Ca2+ binds to a second, lower affinity site on the enzyme. This latter binding influences the interaction of phospholipase A2 with lipid-water interfaces by shifting the pK of the a-NH3+ group of the N-terminal Ala residue from 8.4 to 9.3 [Slotboom, A. J., Jansen, E. H. J. M., Vlijm, H., Pattus, F., Soares De Araujo, P., & De Haas, G. H. (1978) Biochemistry 17, 4593-4600], The effects of Ca2+ ion and pH on the pre-steady-state kinetics and on the activity of porcine phospholipase A2 acting on lecithin monolayers were investigated. At basic pH there is a profound decrease of the penetration capacity of the enzyme in the lipid interface as shown by the increase of the induction time of the kinetics. This increase of the induction time with pH is shifted to a higher pH value by increasing Ca2+ ion concentration. There is a good correlation between the change in the pK of the NH3+ of Ala1 and the effect seen in monolayer studies. The surface concentration of the enzyme is independent of pH until the pH region where deprotonation of the α-NH3+ group of Ala1 occurs and then decreases. At pH 6, a further lowering of Ca2+ ion concentration below the saturation of the high-affinity site of phospholipase A2 decreases the penetration rate of the enzyme to the lipid interface without changing its equilibrium surface concentration. This indicates an effect of the catalytic Ca2+ binding site on the activation energy of the binding process. At pH 9, Ca2+ ions bound to the low-affinity site are required for the binding to interfaces. At this pH, removal of Ca2+ ions from the subphase produces a fast expulsion of the enzyme from the interface, From Lineweaver-Burk plots of the specific activity of the enzyme on substrate monolayers as function of Ca2+ ion concentration, it was concluded that the low-affinity site not only is indispensable for the penetration at basic pH but also affects the turnover of the enzyme. © 1979, American Chemical Society. All rights reserved.