DETERMINANTS OF FUNCTIONAL DIVERSITY IN HEME ENZYMES

人们对生物科学的许多领域都有浓厚的兴趣 在发展我们改变或“改造”蛋白质的能力方面， 人工合成的酶。 然而，我们经常发现， 试图改变我们没有完全改变的结构， 即使蛋白质的结构 是众所周知的。 在蛋白质操作能够合理地 被称为蛋白质工程，有必要实现 在一组高度相关的蛋白质中， 以及催化位点周围的氨基酸 有助于区分一种活动和 另 这些研究的长期目标是能够将 一种血红素过氧化物酶的性质与另一种的性质， 底物特异性的改变，或对另一类底物特异性的改变 血红素蛋白质如肌红蛋白的改变， 血红素活性位点的环境。 然而，许多 影响血红素酶功能的结构因素仍然是 有争议，需要更仔细的检查。 因此 这项建议的具体目的是为了更好地了解 蛋白质之间的相互作用决定了 这些蛋白质在原子水平上。 的变体形式的功能和光谱性质 细胞色素c过氧化物酶和辣根过氧化物酶， 将检查定点诱变以解决 以下问题。 1. 细胞色素c过氧化物酶活性位点通道的突变 具有明显不同功能的这些功能将由低 温度电子顺磁共振 这将提供 Fe+3的低激发态的能量分裂，以及 应该是一个非常敏感的方法来探索环境和 突变对Fe+3中心的构象影响。 2. 位于细胞色素c 过氧化物酶-细胞色素C复合物将被改变。 电子转移 测量将被检查，以确定有效的标准， 电子转移 3. 突变改变的还原电位的研究 细胞色素c过氧化物酶可以决定是否非常消极的 观察到的过氧化物酶的电位是由于单一因素，或 蛋白质环境效应的结合。 4. 含特异性同位素的细胞色素c过氧化物酶的研究 替换将扩大对稳定的位置的搜索 自由基化合物I中间体。 此外， 活性位点附近的氨基酸残基起控制作用， 血红素铁和自由基位点之间的相互作用将 追究 5. 辣根过氧化物酶C的基因将被合成， 在大肠杆菌中表达大肠杆菌进行比较诱变研究， 细胞色素c过氧化物酶的研究进展

There is a significant interest in many areas of biological science in developing our abilities to alter or "engineer" proteins aNd enzymes by artificial means. It is often found, however, that we are attempting to modify structures that we do not fully understand, even for cases in which the structure of the protein is precisely known. Before protein manipulation can be reasonably referred to as protein engineering, it is necessary to achieve an understanding, within a group of highly related proteins, of how and to what degree the amino acids surrounding a catalytic site contribute to the properties that distinguish one activity from another. The long term goal of these studies is to be able to convert the properties of one of the heme peroxidases to those of another by alteration of substrate specificities, or to those of another class of heme protein such as myoglobin by alteration of the protein environment of the heme active site. However, many of the structural factors that influence heme enzyme function are still controversial and require closer examination. Therefore, the specific aims of this proposal are to gain a better understanding of the protein interactions that determine functional diversity in these proteins at the atomic level. Functional and spectroscopic properties of variant forms of cytochrome c peroxidase and horseradish peroxidase produced by site-directed mutagenesis will be examined in order to address the following issues. 1. Mutations of cytochrome c peroxidase in the active site channel that have markedly different function will be examined by low temperature electron paramagnetic resonance. This will provide the energy splittings of the low-lying excited states of the Fe+3, and should be a very sensitive method to explore the environmental and conformational effects of mutations on the Fe+3 center. 2. Specific residues positioned between the cytochrome c peroxidase-cytochrome c complex will be altered. Electron transfer measurements will be examined to determine criteria for efficient electron transfer. 3. Studies of the reduction potential of mutationally altered cytochrome c peroxidases may determine whether the very negative potentials observed for peroxidases are due to a single factor, or to a combination of protein environment effects. 4. Studies of cytochrome c peroxidase containing specific isotopic substitutions will broaden a search for the location of the stable free radical compound I intermediate. In addition, the role that amino acid residues near the active site play a controlling the interaction between the heme iron and the free radical site will be investigated. 5. The gene for horseradish peroxidase C will be synthesized and expressed in E. coli to allow comparative mutagenesis studies now in progress with cytochrome c peroxidase.