DETERMINANTS OF FUNCTIONAL DIVERSITY IN HEME ENZYMES

人们对生物科学的许多领域都很感兴趣。 在发展我们改变或“设计”蛋白质和 人工合成的酶。然而，人们经常发现，我们 正试图修改我们没有完全修改的结构 理解，即使在蛋白质的结构 是确切地知道的。在对蛋白质进行合理的操作之前 称为蛋白质工程，有必要实现一种 在一组高度相关的蛋白质中理解如何 以及催化部位周围的氨基酸含量有多高 有助于区分一项活动和 又一个。 这些研究的长期目标是能够将 一种血红素过氧化物酶与另一种的性质 改变底物特性，或改变另一类底物特性 通过蛋白质的改变而改变血红素蛋白，如肌红蛋白 血红素活性部位的环境。然而，许多 影响血红素酶功能的结构性因素仍然存在 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 决定细胞功能多样性的蛋白质相互作用 这些蛋白质在原子水平上。 不同构型化合物的功能和光谱性质 产生细胞色素C过氧化物酶和辣根过氧化物酶 将研究定点突变，以解决 以下是问题。 1.细胞色素c过氧化物酶活性部位通道突变 有明显不同功能的人将被LOW检查 温度电子顺磁共振。这将提供 Fe+3低激发态的能量分裂，以及 应该是一种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之间的特定残基 过氧化物酶-细胞色素c复合体将发生改变。电子转移 将对测量进行检查，以确定有效率的标准 电子转移。 3.突变变异体的还原潜力研究 细胞色素c过氧化物酶可能决定非常阴性的 观察到的过氧化物酶电位是由单一因素引起的，或者 对蛋白质环境的综合影响。 4.含特定同位素的细胞色素c过氧化物酶的研究 替换将扩大对马厩位置的搜索 自由基化合物I中间体。此外，这个角色， 活性部位附近的氨基酸残基起着控制作用。 血红素铁和自由基位的相互作用将 被调查。 5.将合成辣根过氧化物酶C基因，并 在大肠杆菌中表达，现在可以进行比较诱变研究 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.