Thermodynamic Reciprocity of the Inhibitor Binding to the Active Site and the Interface Binding Region of IB Phospholipase A2

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Abstract

Interfacial activation of pig pancreatic IB phospholipase A(2) (PLA2) is modeled in terms of the three discrete premicellar complexes (E(i)(#), i = 1, 2, or 3) consecutively formed by the cooperative binding of a monodisperse amphiphile to the i-face (the interface binding region of the enzyme) without or with an occupied active site. Monodisperse PCU, the sn-2-amide analogue of the zwitterionic substrate, is a competitive inhibitor. PCU cooperatively binds to the i-face to form premicellar complexes (E(i), i = 1 or 2) and also binds to the active site of the premicellar complexes in the presence of calcium. In the E(i)I complex formed in the presence of PCU and calcium, one inhibitor molecule is bound to the active site and a number of others are bound to the i-face. The properties of the E(i) complexes with PCU are qualitatively similar to those of E(i)(#) formed with decylsulfate. Decylsulfate binds to the i-face but does not bind to the active site in the presence of calcium, nor does it interfere with the binding of PCU to the active site in the premicellar complexes. Due to the strong coupling between binding at the i-face and at the active site, it is difficult to estimate the primary binding constants for each site in these complexes. A model is developed that incorporates the above boundary conditions in relation to a detailed balance between the complexes. A key result is that a modest effect on cooperative amphiphile binding corresponds to a large change in the affinity of the inhibitor for the active site. We suggest that besides the binding to the active site, PCU also binds to another site and that full activation requires additional amphiphiles on the i-face. Thus, the activation of the inhibitor binding to the active site of the E(2)(#) complex or, equivalently, the shift in the E(1)(#) to E(2)(#) equilibrium by the inhibitor is analogous to the allosteric activation of the substrate binding to the enzyme bound to the interface.

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