Binding of porcine pancreatic phospholipase A2 to various micellar substrate analogues. Involvement of histidine-48 and aspartic acid-49 in the binding process.

Abstract

The interaction of porcine pancreatic phospholipase A2 (PA2) with micelles of various single-chain phospholipid analogues was studied by ultraviolet absorption difference spectroscopy and light-scattering measurements. The phospholipids used were either substrate analogues or products, varying in hydrocarbon chain lengths and polar head groups. The results indicate that the enzyme forms a stable complex over a wide range of enzyme and lipid concentrations. From the equivalent "molecular weight" and from the lipid to enzyme molar ratio (N) of the micelle--enzyme complex, it can be calculated that complexes containing saturated hydrocarbon chain lipids generally consist of two enzyme molecules and half of the number of lipid monomers present in free micelles. The interaction forces between the enzyme and lipid monomers bound in the complex are mainly hydrophobic. Stronger binding is found when the essential cofactor Ca2+ is bound to the enzyme. pH-titration studies on the binding of native PA2 to aggregated lipid structures showed that at least one group with a pKA value of 6.25 is involved in the interaction with lipid micelles. At acidic pH, micelle binding is stronger than at neutral or alkaline pH. Alkylation of the active site residue His48 resulted in a shift of the pKA value to 4.6, while addition of Ca2+ appears to stabilize the micelle-binding conformation of both native and modified enzymes over a broad pH range (pH 4--9.5). From these observations it is suggested that both the Ca2+ binding residue Asp49 [Fleer, E. A. M., Verheij, H. M., & de Haas, G. H. (1980) Eur. J. Biochem. 113, 283--288] and His48 control micelle binding of the native enzyme. For optimal binding in the absence of Ca2+, a long-distance hydrogen bond between these two residues is required; this can be established via a water molecule. It is assumed that it is a proton of this "H bond" which is titrated with a pKA value of 6.25. When the "H bond" is absent, as in the alkylated enzymes, Asp49 alone controls micelle binding with a pKA of 4.6. These results, together with the effect of Ca2+ on micelle binding, indicate that it is not the "hydrogen bridge" between His48 and Asp49 which is of main importance for an optimum binding conformation of the enzyme but the effective charge in the microenvironment of Asp49. It is proposed that a negative charge on this carboxylate causes a conformational change of the enzyme which leads to a protein conformation lacking an active micelle binding site. Binding of Ca2+ or reprotonation neutralizes this negative charge and restores the enzyme's ability to bind micelles.