METALLOENZYME MECHANISMS

通过本申请中定义的研究计划，我们寻求 了解金属酶功能的分子机制， 金属配位与各种化合物比活度的关系 金属蛋白 我们的第一项工作将集中在氧结合 和代谢蛋白质血红蛋白、肌红蛋白、过氧化物酶和细胞色素 P-450 具有金属辅因子的蛋白质系统在哺乳动物中普遍存在。 大多数活组织的基本生物转化，并与 许多明确的疾病状态。 在许多情况下，金属中心提供 由金属配位决定的独特催化活性 球体。 我们建议研究金属配体的确切作用， 通过使用位点特异性诱变的各种催化过程， 其他现代重组DNA技术来特异性地改变这种连接 球体。 然后，我们将确定这些修改对 金属蛋白的结构和活性。 总体目标是 分离那些仅仅由于 配位配体和那些由蛋白质结构决定的配体 和环境 从这些调查中出现的将是第一个 金属配位在生物学中的作用的明确文件 一组重要的金属蛋白系统的活性。 我们的第一 具体的目标将集中在细胞色素P-450单加氧酶系统发现 在自然界中普遍存在，并在解毒，异生物质 代谢和类固醇生物合成。 细胞色素P-450具有 与未活化烷烃相关的独特化学反应性 羟基化，且还含有同样不常见硫醇盐轴向配体 到血红素中心 我们希望确定这种半胱氨酸的功能 配体在决定相关的化学反应性和生物物理 P-450加氧酶的性质。 重组DNA技术将是 用于将细胞色素P-450中的血红素轴向配体从半胱氨酸改变为 组氨酸侧链更常见于肌红蛋白、血红蛋白和 过氧化物酶。 匡威的实验将取代组氨酸残基， 过氧化物酶和具有硫醇盐的血红蛋白/肌红蛋白。 通过应用 EPR、ENDOR、穆斯堡尔、拉曼、MCD和X射线结构测定， 沿着快速反应和精确的化学反应性记录， 可以清楚地分离轴向配体的贡献， 特定的活性位点群和蛋白质环境中的独特电子 与P-450血红素中心相关的转移和氧化学。

Through the research program defined in this application, we seek to understand the molecular mechanisms of metalloenzyme function and the relationship of metal coordination to the specific activities of various metalloproteins. Our first efforts will be focused on the oxygen binding and metabollic proteins hemoglobin, myoglobin, peroxidase, and cytochrome P-450. Protein systems with metal cofactors are ubiquitous in the essential biotransformations of most living tissues and are associated with many well defined disease states. In many cases, the metal center offers a unique catalytic activity which is dictated by the metal coordination sphere. We propose to investigate the precise role of the metal ligands in various catalytic process through the use of site specific mutagenesis and other modern recombinant DNA techniques to specifically alter this ligation sphere. We will then determine the effects of such modifications on the structure and activity of the metalloprotein. The overall goal is to separate those features and reactivities which are due solely to the coordinating ligands and those which are dictated by the protein structure and environment. Emerging from these investigations will be the first clear documentation of the role of metal coordination in the biological activity of an important group of metalloprotein systems. Our first specific aim will focus on the cytochrome P-450 monoxygenase systems found ubiquitously in nature and playing key roles in detoxification, xenobiotic metabolism, and steroid biosynthesis. The cytochromes P-450 possess the unique chemical reactivities associated with unactivated alkane hydroxylation, and also contain a likewise uncommon thiolate axial ligand to the heme center. We wish to determine the function of this cysteine ligand in dictating the relevant chemical reactivities and biophysical properties of the P-450 oxygenases. Recombinant DNA technology will be used to change the heme axial ligand in cytochrome P-450 from cysteine to the histidine side chain more commonly found in myoglobin, hemoglobin, and peroxidase. The converse experiment will replace the histidine residue in peroxidase and hemoglobin/myoglobin with a thiolate. Through application of EPR, ENDOR, Mossbauer, Raman, MCD and X-ray structure determination, along with rapid reaction and precise chemical reactivity documentations, it will be possible to clearly separate the contributions of axial ligand, specific active site groups, and protein environment to the unique electron transfer and oxygen chemistries associated with the P-450 heme center.