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和金属酶 金属配位与各种不同配位的特定活性的关系 金属蛋白。我们的第一个努力将集中在氧结合上 以及代谢蛋白血红蛋白、肌红蛋白、过氧化物酶和细胞色素 P-450。含有金属辅因子的蛋白质系统普遍存在于 大多数活组织的基本生物转化，并与 许多定义明确的疾病状态。在许多情况下，金属中心提供了一个 由金属配位决定的独特的催化活性 球体。我们建议研究金属配体在体内的确切作用。 各种催化过程通过使用定点突变和 其他现代重组DNA技术可以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 分离那些完全由 配位配体和由蛋白质结构决定的配位配体 和环境。从这些调查中出现的将是第一个 清楚地记录金属配位在生物中的作用 一组重要的金属蛋白系统的活性。我们的第一次 具体目标将集中在发现的细胞色素P-450单加氧酶系统上 在自然界中无处不在，并在解毒、异种生物 新陈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 羟基化，还含有同样罕见的硫代轴向配体 去亚铁血红素中心。我们希望确定这种半胱氨酸的功能 配体在决定相关化学反应和生物物理活性中的作用 P-450加氧酶的性质。重组DNA技术将成为 用于将细胞色素P-450中的血红素轴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。反向实验将取代组氨酸残基在 过氧化物酶和血红蛋白/肌红蛋白与硫酸盐。通过应用程序 EPR、Endor、穆斯堡尔、拉曼、MCD和X射线结构测定， 伴随着快速反应和精确的化学反应记录， 将有可能清楚地分离轴向配体的贡献， 特定的活性位点群，以及蛋白质环境中的独特电子 与P-450血红素中心有关的转移和氧化学。

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.