TERMINAL ENZYMES OF HEME SYNTHESIS

本研究计划的长期目标是通过基本的生物化学、结构和分子生物学方法来了解这些蛋白质的结构/功能关系，了解该途径末端酶的正常催化功能。收集到的数据已经并将继续使人们更好地了解卟啉症的生化性质，并可能为这些疾病的可变外显率提供一些线索。这些实验还帮助我们了解血红素生物合成酶的催化功能，以便人们可以更好地预测或解释某些药物治疗方案的潜在负面影响。在下一个资助期，我们计划继续我们对铁螯合酶和原卟啉原氧化酶的结构/功能研究，并将我们的研究扩大到包括假定的哺乳动物细胞内血红素结合蛋白，因为它可能在细胞内血红素（卟啉）的运动、储存和/或调节中发挥重要作用。我们建议在下一个拨款期着手处理的具体事项如下：1.)鉴定位于铁螯合酶活性位点内参与底物结合和催化的残基，并表征它们的特定作用，2.)确定（2Fe-2S]簇对拥有它的铁螯合酶的作用，3.）检查二聚化对铁螯合酶和原卟啉原氧化酶的作用，并确定单个亚基是否独立起作用。4.真核生物铁螯合酶的膜结合机制表征及其体内必要性的确定，5.人原卟啉原氧化酶活性位点残基的鉴定及其催化作用的表征，6.原卟啉原氧化酶催化机制的进一步表征。7.)原卟啉原氧化酶的结晶和8.)哺乳动物细胞内血红素结合蛋白的表征和结构测定。位点导向突变体和野生型酶的研究将采用多种生物物理技术，这些技术以前在我们的实验室和我们的合作者的实验室中使用过。

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.