RELATIONSHIP OF INTERFACIAL EQUILIBRIA TO INTERFACIAL ACTIVATION OF PHOSPHOLIPASE-A(2)

The equilibrium dissociation constants for the distribution of pig pancreatic phospholipase A2, its competitive inhibitors, and their complexes at the interface of a neutral diluent are determined. The relationship between these parameters and their significance for interfacial catalysis is elaborated in terms of a model based on the relationship between the underlying equilibria. By using a combination of spectroscopic and chemical modification methods, it was possible to determine the equilibrium dissociation constant of an inhibitor bound to the interface (K') or of the inhibitor bound to the enzyme in the aqueous phase (K(I)) or the interface (K(I)). The equilibrium dissociation constant for the free enzyme (K(d)) or for the enzyme-inhibitor complex (K(d)I) from the interface were also obtained. These constants are shown to be thermodynamically related, i.e., K'K(d)K(I) = K(I)K(d)I, as predicted on the basis of the cyclic equilibrium scheme (thermodynamic box) describing the distribution of the enzyme and the inhibitor between the aqueous phase and the interface at constant calcium concentration. Results show that (i) calcium is required for the binding of a substrate or inhibitor molecule to the catalytic site; (ii) the effective dissociation constant of the inhibitor-enzyme complex in the aqueous phase is considerably larger than that for the enzyme at the interface, i.e., K(I) >> effective K(I); (iii) K(d)I does not depend on the structure of the inhibitor and K(d) >> K(d)I; and (iv) structure-activity correlations suggest that ionic interactions between a ligand and the interfacial recognition site of the enzyme are important for K', which controls the concentration of the bound inhibitor that the enzyme ''sees'' in the interface. These observations demonstrate that the binding of the enzyme to the interface and the binding of the inhibitor to the active site of the enzyme at the interface are two distinguishable processes. Therefore, binding of a ligand to the active site of the enzyme promotes binding of the enzyme-inhibitor complex to other amphiphiles and the interface with higher affinity. It is suggested that the primary effect of binding the enzyme to the interface is to increase its intrinsic affinity toward the active-site-directed ligands, i.e., the interfacial activation of PLA2 is of K-type.