The two long range objectives of this program involve mechanistic studies on peripheral membrane enzymes and hemoproteins. One objective is to define the molecular mechanisms involved in the activation and regulation of pyruvate oxidase, a peripheral membrane protein whose catalytic activity (kcat/Km) is increased approximately 450-fold by binding to membrane vesicles. Pyruvate oxidase also is activated by bonding to a wide variety of natural and synthetic 1ipid amphiphiles or by the cleavage of a 23 amino acid residue peptide (a-peptide) from the carboxy terminal domain of the enzyme. The immediate goals for the pyruvate oxidase project involve: 1) Identification of essential amino acid residues in the a-peptide, the putative membrane binding domain in pyruvate oxidase; 2) Characterization of hinge mutants; 3) Identification of the rate limit step which responds to lipid and protease activation, 4) X-ray crystal structure of activated and inactivated enzyme. A second objective of this program involves hemoprotein structure- function studies primarily centered on the chemistry of peroxidase, most notable the relation of cholorperoxidase to plant peroxidase, catalase, and P-450 cytochromes. Cholorperoxidase is unique in that in addition to catalyzing peroxidative nalogenation catalase and P-450 cytochromes. The immediate goals of the hemoprotein project include: 1) The three dimensional structure of choloroperoxidase are deduced by X-ray crystallographic analysis; 2) Mutagenesis of specific active site residues; 3) The chemical characterization of intermediates in peroxidase, catalase, and P- 450 reactions; 4) The mechanism of the halide dependent activation of chloroperoxidase.
