The objective of this project is to study the quantitative structure-function relationship of phospholipase A2 (PLA2) by a combination of genetic, biochemical, bioorganic, and biophysical techniques. The system will be the PLA2 from bovine pancreas, expressed in E. coli by the synthetic gene approach.
In Specific Aim 1, we will use site-specific mutagenesis to identify the residues involved in stabilization of the transition state. This will state with a """"""""working"""""""" of the PLA2-substrate complex. The focus will be on a potential H-bonding to the phosphate (possibly through Tyr-69), on the catalytic role of the """"""""catalytic diad"""""""" Asp- 99...His-48 and its resemblance (or lack of) to serine proteases, and on the hydrophobic interaction with the acyl chains of the substrate. The roles of the absolutely conserved Tyr-52 and Tyr- 73 will be tested.
In Specific Aim 2, we will use site-specific mutagenesis to determine the structural and functional roles of several highly conserved hydrophobic residues (lle-9, Phe-5, 22, 106, Ala-102, 103, and a disulfide linkage between Cys-29 and Cys- 45) surrounding the inner cleft of the active site. The """"""""site- specific mutagenesis"""""""" in Aims 1 and 2 involves six major steps including characterization of the kinetic and (qualitative) structural properties of each mutant.
In Specific Aim 3, the level of expression and the yield of purification and renaturation of PLA2 will be improved to a level suitable for biophysical studies (>or equal to mg/liter).
In Specific Aim 4, detailed proton NMR (1D and 2D) and C-13 NMR 1D) analysis will be conducted on two systems: (a) For the mutants showing substantially perturbed kinetic or structural properties (judging from kinetic assays, 1D proton NMR, and conformational and thermal stability to be measured in Specific Aims 1 and 2), we will compare their conformations with the wild-type (WT) conformation. (b) For WT and selective mutants, we will use 1D and 2D proton NMR to compare the structure of PLA2 in solution with its complexes with tight-binding inhibitors (potentially transition state analogues). The studies with WT will state immediately using the enzyme purified from bovine pancreas.
In Specific Aim 5, we will produce """"""""hybrid enzymes"""""""" to test the structural-functional comparison between bovine pancreatic PLA2 (monomer) and C. atrox venom PLA2 (dimer, Kd 10-9-10-11M) suggested on the basis of detailed analysis of their crystal structures. These two enzymes are ca. 50% homologous and represent each of two """"""""distant groups"""""""" of PLA2. Their backbone conformation ar largely superimposable except three regions. We will modify the pancreatic PLA2 to mimic the C. atrox PLA2 in each of the three regions and examine its effect of structure and function. In addition, we will express the c. atrox PLA2 and perform complementary studies. The information to be generated will be useful in the design of drugs (enzyme inhibitors) and artificial enzymes.
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