METALLOENZYME MECHANISMS

我们将探讨金属酶催化的分子机制 通过使用重组DNA方法。 我们力求提供 在大分子识别方面， 碳链官能化的区域和立体特异性， 氧化催化的化学机理和金属中心的作用 配体在确定血红素的电子和光谱性质中的作用 和铁硫中心。 具体来说，我们感兴趣的是 通过这种方法，大分子既能识别其小分子底物， 以及形成金属酶复合物所必需的辅助蛋白质， 精确定义的拓扑结构细胞色素P-450 cam，细胞色素b5， 细胞色素C和肌红蛋白，其中在所有情况下， 已知结构具有高分辨率。 蛋白质-蛋白质识别是 通过表面电荷诱变和高压光谱研究。 我们 正在使用定点突变来研究双原子的机制， 配体识别，允许氧的运输，储存， 呼吸作用发生在正常生理水平的碳 一氧化物的产生，否则会毒害血红素蛋白质。 的 特异性，无论是在区域和立体选择性，细胞色素 P-450 cam也被作为分子识别的问题进行研究， 我们的初步结果显示， 酶活性位点的工程改造，用于催化的从头设计 处理. 我们继续工作的最后一个主要具体目标是 描述催化的化学机制，如由特定的 活性位点中单个氨基酸侧链的需求 环境 例如，通过改变金属中心配体 （组氨酸，酪氨酸和半胱氨酸），我们已经能够产生新的 金属蛋白的催化活性。 提供新的活性部位 酸碱功能可以为开发更多的 高效和新颖的催化剂。 芳香族氨基酸的诱变作用是 用于定义依赖于路径的电子转移反应。 在 总之，GM 33775带来了强大的重组DNA技术， 技术承担的重要问题，金属酶机制。

We will investigate the molecular mechanisms of metalloenzyme catalysis through the use of recombinant DNA methodology. We seek to provide the structure-function link in aspects of macromolecular recognition, the regio- and stereospecificity of carbon chain functionalization, the chemical mechanisms of oxidative catalysis, and the role of metal center ligands in determining the electronic and spectroscopic properties of heme and iron-sulfur centers. Specifically, we are interested in the mechanisms by which a macromolecule recognizes both its small molecule substrate as well as the ancillary proteins necessary to form metalloenzyme complexes of the precisely defined topology cytochrome P-450cam, cytochrome b5, cytochrome c, and myoglobin where in all cases the three dimensional x-ray structures are known to high resolution. Protein-protein recognition is studied by surface charge mutagenesis and high pressure spectroscopy. We are using site directed mutagenesis to examine the mechanisms of diatomic ligand discrimination that allows oxygen transport, storage, and respiration to occur under the normal physiological levels of carbon monoxide production which would otherwise poison heme proteins. The specificity, both in terms of regio- and stereoselectivity, of cytochrome P-450cam is also being examined as a question of molecular recognition, and our initial results have indicated the feasibility of complete re- engineering of an enzyme active site for de novo design of catalytic processing. A final major specific aim of our continuing work is to delineate the chemical mechanisms of catalysis as dictated by the specific requirement of individual amino acid side chains in the active site environment. For example, by alteration of metal center ligands (histidine, tyrosine, and cysteine) we have been able to generate new catalytic activities of metalloproteins. Provision of new active site acid-base functions can open the possibility for development of more efficient and novel catalysts. Mutagenesis of aromatic amino acids is being used to define path-dependent electron transfer reactions. In summary, GM33775 brings the powerful techniques of recombinant DNA technology to bear on the important problems in metalloenzyme mechanisms.