Structural basis of the anionic interface preference and k*(cat) activation of pancreatic phospholipase A(2)

Pancreatic phospholipase A(2) (PLA2) shows a strong preference for the bind ing to the anionic interface and a consequent allosteric k(cat)*, activatio n. In this paper, we show that virtually all the preference is mediated thr ough 3 (Lys-53, -56, and -120) of the 12 cationic residues of bovine pancre atic PLA2, The lysine-to-methionine substitution enhances the binding of th e enzyme to the zwitterionic interface. and k(cat)* for the K53,55,120M tri ple mutant at the zwitterionic interface is comparable to that for the wild type (WT) at the anionic interface. In the isomorphous crystal structure, the backbone folding of K53,56M K120,121A and WT are virtually identical, y et a significant change in the side chains of certain residues, away from t he site of substitution, mostly at the putative contact site with the inter face (i-face), is discernible, Such reciprocity, also supported by the spec troscopic results for the free and bound forms of the enzyme, is expected b ecause a distal structural change that perturbs the interfacial binding cou ld also affect the i-face. The results show that lysine-to-methionine subst itution induces a structural change that promotes the binding of PLA2 to th e interface as well as the substrate binding to the enzyme at the interface . The kinetic results are consistent with a model in which the interfacial Michaelis complex exists in two forms, and the complex that undergoes the c hemical step is formed by the charge compensation of Lys-53 and -56, Analys is of the incremental changes in the kinetic parameters shows that the char ge compensation of Lys-53 and -56 contributes to the k(cat)*, activation an d that of Lys-120 contributes only to the structural change that promotes t he stability of the Michaelis complex at the interface. The charge compensa tion effects on these three residues also account for the differences in th e anionic interface preference of the evolutionarily divergent secreted PLA 2.