In this competitive renewal, Dr. Dailey plans to continue his in depth investigations on ferrochelatase, protoporphyrinogen oxidase and on the role of the mitochondrial membrane in heme biosynthesis. In addition, his field of research will be expanded to coproporphyrinogen oxidase so that the terminal three enzymes of the heme biosynthetic pathway will be covered in this project. These enzymes are of general biochemical interest not only for their unique catalytic functions, but also because of their vectorial organization across the membrane. Medically they are of importance since decreased activities of any one of them results in a disease condition known generally as porphyria. In addition, drug induced heme destruction in cytochrome P450 can lead to a porphyric condition in healthy animals, and it appears that some anemias may result from disordered heme synthesis.
The research aims of this proposal are to investigate the structure-function relationships of these three enzymes to gain a better understanding of how each carries out its catalytic function in normal cells. Specifically, the applicant proposes to examine structural features of the three enzymes making use of circular dichroism in conjunction with limited proteolysis, binding to phospholipid vesicles, and chemical modifications. These data are expected to show something about the overall secondary structure of each of these enzymes and what role membrane association may play in stabilizing a structural motif. One of the major goals of this proposal is to investigate structure-function relationships by producing altered enzymes via PCR-mediated site directed mutagenesis. The applicant is planning to initially concentrate on the generation and characterization of mutants of murine ferrochelatase. Later, after the cDNA for PPO and CPO have been isolated and expressed, Dr. Dailey proposes to address these same topics with those enzymes. Of particular interest will be the identification of the iron binding residues and the putative porphyrin aligning arginyl residue(s) of ferrochelatase. Modified forms of ferrochelatase will be constructed so that the enzyme can be targeted to different intracellular locations. The applicant plans to conduct these studies in an effort to determine what role its position on the matrix side of the inner mitochondrial membrane has in its activity in vivo. Data gathered in these experiments are expected to help in understanding the nature of the catalytic features of these enzyme. They are also expected to help in explaining the nature of the defect in the relevant porphyria and may be of value in evaluating some drug and chemical induced porphyrinopathies.
Showing the most recent 10 out of 64 publications
