Structural Basis of the Anionic Interface Preference and k cat / Activation of Pancreatic Phospholipase A 2 ² Bao-Zhu Yu, ‡ Ming Jye Poi, § U. A. Ramagopal, # Rinku Jain, # S. Ramakumar, # Otto G. Berg,* ,⊥ Ming-Daw Tsai,* ,§ K. Sekar,* ,# and Mahendra Kumar Jain* ,‡ Department of Chemistry and Biochemistry, UniVersity of Delaware, Newark, Delaware 19716, Department of Physics and Bioinformatics Centre, Indian Institute of Science, Bangalore, India, Departments of Chemistry and Biochemistry and Ohio State Biochemistry Program, The Ohio State UniVersity, Columbus, Ohio 43210, and Department of Molecular EVolution, Uppsala UniVersity EVolutionary Biology Center, Uppsala, Sweden ReceiVed March 31, 2000; ReVised Manuscript ReceiVed July 24, 2000 ABSTRACT: Pancreatic phospholipase A 2 (PLA2) shows a strong preference for the binding to the anionic interface and a consequent allosteric k cat / activation. In this paper, we show that virtually all the preference is mediated through 3 (Lys-53, -56, and -120) of the 12 cationic residues of bovine pancreatic PLA2. The lysine-to-methionine substitution enhances the binding of the enzyme to the zwitterionic interface, and k cat / for the K53,56,120M triple 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, yet a significant change in the side chains of certain residues, away from the site of substitution, mostly at the putative contact site with the interface (i-face), is discernible. Such reciprocity, also supported by the spectroscopic results for the free and bound forms of the enzyme, is expected because a distal structural change that perturbs the interfacial binding could also affect the i-face. The results show that lysine-to-methionine substitution induces a structural change that promotes the binding of PLA2 to the 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 chemical step is formed by the charge compensation of Lys-53 and -56. Analysis of the incremental changes in the kinetic parameters shows that the charge compensation of Lys-53 and -56 contributes to the k cat / activation and that of Lys-120 contributes only to the structural change that promotes the stability of the Michaelis complex at the interface. The charge compensation effects on these three residues also account for the differences in the anionic interface preference of the evolutionarily divergent secreted PLA2. Regulation of the interfacial catalytic behavior of an enzyme by the interface is not adequately understood, and the structural basis for the interface preference is one of the major unresolved problems in interfacial enzymology. For a general formulation of the consequences of interfacial catalysis and activation, we have characterized the catalytic behavior of pancreatic phospholipase A 2 (PLA2) 1 in terms of the primary interfacial rate and equilibrium parameters (1-10). Significantly higher rates of hydrolysis of PLA2 at anionic interfaces, relative to those at zwitterionic interfaces, have been dissected into enhanced binding of PLA2 (4, 6, 7) and the allosteric k cat / activation for the turnover at the interface (6, 8, 9). Such a preference for the anionic interface, attributed to a change in the intrinsic properties of the enzyme at the interface, is clearly discernible from the apparent rate enhancement due to an increased substrate replenishment in the microenvironment of the enzyme at the interface (10- 13). Having resolved the functional consequences of the anionic interface preference in terms of the primary parameters, in this paper we identify the cationic residues that contribute to the anionic interface preference of bovine pancreatic PLA2. Since the natural substrate of pancreatic PLA2 is codispersed with anionic bile salts, a role for cationic residues has been widely recognized (9, 14-20). The anionic interface preference is a general property shared to varying degrees by several evolutionarily divergent secreted PLA2 isoforms and other interfacial enzymes. Our earlier analysis of the kinetic effects of the single lysine substitutions (9) and deletions (20) in bovine PLA2 had suggested an incremental role for Lys-53, -56, -120, and -121 in the anionic interface ² This work was supported by PHS (GM29703 to M.K.J., GM41788 to M.D.T.), Swedish NSRC (O.G.B.). * To whom correspondence should be sent. Phone: 302-831-2968; fax: 302-831-6335; e-mail: mkjain@udel.edu. ‡ University of Delaware. § Ohio State University. # Indian Institute of Science. ⊥ Uppsala University. 1 Abbreviations: CMC, critical micelle concentration; DC7PC, 1,2- diheptanoylglycero-sn-3-phosphocholine; deoxy-LPC, 1-hexadecylpro- panediol-3-phosphocholine; DMPC, dimyristoyl-sn-3-glycerophospho- choline; DOPC, dioleoyl-sn-3-glycerophosphocholine; i-face, the interfacial contact face of PLA2; MJ33, 1-hexadecyl-3-(trifluoroethyl)- rac-glycero-2-phosphomethanol; PLA2, phospholipase A 2 from bovine pancreas (nmPLA2, isozymes from Naja melanoleuca venom; app- PLA2, the monomer isozyme from Agkistrodon piscoVorus piscoVorus venom); WT, bovine pancreatic PLA2. 12312 Biochemistry 2000, 39, 12312-12323 10.1021/bi000740k CCC: $19.00 © 2000 American Chemical Society Published on Web 09/08/2000