TERMINAL ENZYMES OF HEME SYNTHESIS

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The long term goal of this research program is to understand the normal catalytic functioning of the terminal enzymes of the pathway via basic biochemical, structural, and molecular biological approaches to structure/function relationships of these proteins. Data collected have and will continue to yield a better understanding of the biochemical nature of porphyrias and may provide some clues about the variable penetrance of these disorders. These experiments also help us to understand the catalytic functioning of the heme biosynthetic enzymes so that one might better anticipate or explain the potential negative aspects of certain drug therapy regimes. In this next grant period we propose to continue our structure/function studies on ferrochelatase and protoporphyrinogen oxidase and will expand our studies to include the putative intracellular mammalian heme binding protein since it may play an important role in intracellular heme (porphyrin) movement, storage, and/or regulation. Specific points that we propose to approach in this next grant period are: 1.) the identification of those residues located within the active site of ferrochelatase that are involved in substrate binding and catalysis, and the characterization of their specific roles, 2.) determination of the role that the (2Fe-2S] cluster plays for ferrochelatases that possess it, 3.) examination of the role that dimerization plays for both ferrochelatase and protoporphyrinogen oxidase and to determine if the individual subunits function independently. 4.) characterization of the membrane binding mechanism for eucaryotic ferrochelatase and determination of its in vivo necessity, 5.) identification of active site residues of human protoporphyrinogen oxidase and characterization of the roles that these residues play in catalysis, 6.) further characterization of the catalytic mechanism of protoporphyrinogen oxidase, 7.) crystallization of protoporphyrinogen oxidase and 8.) characterization and structure determination of the mammalian intracellular heme binding protein. Studies on site-directed mutants and wildtype enzymes will employ a variety of biophysical techniques that we have utilized previously in our laboratory and the laboratories of our collaborators.