) Many enzymes, including those of lipid metabolism, have evolved to access their substrates at the interface, and processively carry out the catalytic turnover at the interfaces of micelles or bilayer membrane. For example, a dozen different phospholipase A2 (PLA21) in human tissues act on phospholipids of membranes. The products and their eicosanoid metabolites signal and regulate a wide range of signaling, secretory, and inflammatory processes. We have established protocols for the interfacial kinetic analysis of PLA2. The challenge is to dissect the interfacial catalytic turnover events from other processes that influence the microscopic steady state condition for the ensemble-averaging. Dissection of the binding of the enzyme to the interface from the interfacial catalytic turnover events permits determination of the primary rate, equilibrium, and activation parameters. This approach has led to novel insights into the catalytic mechanism. Interfacial activation is dissected as increased substrate affinity of the enzyme at the interface (Ksstar-allostery), and the kcatstar activation is mediated by the anionic interface. Our next focus is to develop a structural basis for the allosteric interfacial activation. Our working model is that pancreatic PLA2 at the interface exists in two forms: the charge-compensated form at the anionic interface is catalytically active (EstarS)#, and the charge-sensitive form at the zwitterionic interface (EstarS) is impaired (Scheme II).
Specific aims of the proposed study include: (#1) To discern the role of Lys-l0 and Lys-62 and (#2) of the 63-66 loop in the PLA2 binding to the interface and the catalytic events. Mutants with a single Trp at 1, 10, 19,20, 31, 53, 56, 62 ,69, 73, 87, 115 or 120 will be prepared with the W3F and with or without the K53,56, 121M substitution to represent the charge-sensitive and the charge-compensated forms of pig pancreatic PLA2 (isoform IB). Both forms of the Trp-mutants will be characterized for (#3) the catalytic, activation, and binding parameters at the anionic versus zwitterionic interfaces, and also (#4) spectroscopically to ascertain the quencher accessibility of the single Trp-probe in different positions. Together, results of aims #1-4 will be used to identify the face of IB PLA2 (the i-face) that makes contact with the interface, and how this face differs at the anionic versus zwitterionic interfaces. (#5) The anion binding sites of PLA2 will be identified from the x-ray structure of the cocrystals with certain anions, such as sulfate and phosphate. (#6) Key results from these aims will be extended to other PLA2 isoforms and their site-directed mutant's. Thus the overall goal is to identify the residues at the i-face, identify the anion binding sites, characterize the tertiary structural and functional differences between the PLA2 isoforms. These results will provide insights into the structural basis for the functional differences between the coupling of the i-face with the active site events of the PLA2 family.
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