The proposed work involves the chemical characterization of enzyme intermediates in oxygen activation and enzymatic halogenation reactions and their relation to peroxidase and cytochrome P-450 chemistry. A major focus of the work centers on the chemical characterization of peroxidase Compound I and Compound X. We also plan to continue our work on the three dimensional structure of chloroperoxidase, especially at the heme active site of this enzyme. Previous work has established a close relation between chloroperoxidase, P-450 cytochromes and model compounds containing a thiolate ligand to heme iron. We plan to continue our work on the characterization of the lipid and proteaseactiviated forms of pyruvate oxidase. The catalytic activity of this peripheral membrane enzyme is dramatically increased by the binding of amphiphilic molecules to a unique high affinity lipid binding site on the enzyme. A covalent affinity label has been developed in order to identify this high affinity binding site. Proteolytic nicking of the native 60 K monomer of pyruvate oxidase produces a 58 K species having the catalytic activity of the lipid activated anzyme. The 58 k species does not require lipid amphiphiles for enhanced activity and in fact has lost the lipid binding site associated with the 60 K monomer.
