STUDIES OF THE DYNAMICS OF HEME PROTEIN ACTIVE SITES

这项提案中描述的工作是我们继续工作的延伸 努力描述和比较两种独特的亚铁血红素的活性部位动力学 蛋白质。其中之一是细胞色素c过氧化物酶，一种铁血红素酶。 从詹姆斯·埃尔曼博士的实验室里的面包酵母中分离出来。这个 其他蛋白质实际上是一组蛋白质，它们被单独地 在我自己的实验室里分离和纯化的，组成了所谓的 二枝甘油的“单体血红蛋白”组分。细胞色素c 过氧化物酶(CCP)已被用不太精确的光谱方法研究， 但我们的初步工作表明，质子核磁共振 光谱学可以有效地用来确定 特定的组，既对蛋白质，又对血红素组，影响 酶的反应性(见下文，另见重印和预印本)。 我们计划用饱和核磁共振继续我们的动力学研究 传输和红场脉冲序列实验。这些将导致 超精细移位质子的进一步归属、血红素结合分析 动力学，解释超精细移位质子的不稳定性和 外在分子间通讯程度的量化 以及作为活性中心的组成部分的单个原子。我们的工作 第二组蛋白质正处于快速膨胀的边缘 由于我们在最近的过去努力净化了 甘油单体组分。这一努力中最重要的是这样一个事实 这些组件中的一个已被确定为缺少远端 组氨酸。对这些蛋白质的研究将包括连接动力学， 平衡配位研究，这些将与光谱相关 结合序列和序列的血红素口袋的特征 在其他地方进行的结构研究。根据这些结论得出的结论 研究将对特定角色的理论产生广泛的影响 哪些末端残基同时在合作和非合作配体中发挥作用 绑定过程。这些将是真正的结构-功能相关性 它可以定义支配主要过程(配体)的特定因素 非共价血红素蛋白的结合)。这项研究的结果将 影响合成的血红素酶和定制的血红素蛋白的设计。

The work described in this proposal is an extension of our continuing effort to describe and compare the active site dynamics of two unique heme proteins. One of these is cytochrome c peroxidase, a ferriheme enzyme isolated from baker's yeast in the laboratory of Dr. James Erman. The other protein is actually a group of proteins which have been individually isolated and purified in my own laboratory and which comprise the so called "Monomer Hemoglobin" fraction of Glycera Dibranchiata. Cytochrome c peroxidase (CcP) has been studied by less precise spectroscopic methods, but our initial work has shown that proton nuclear magnetic resonance spectroscopy can be used effectively to identify the role played by specific groups, both on the protein and on the heme group, which influence the enzyme's reactivity (vide infra, see also the reprints and preprints). We plan to continue our dynamics studies with nmr employing saturation transfer and Redfield pulse sequence experiments. These will result in further assignments of hyperfine shifted protons, analysis of heme binding dynamics, elucidation of the lability of hyperfine shifted protons and quantitation of the extent of communications between extrinsic molecules and the individual atoms which are components of the active site. Our work on the second group of proteins is on the verge of rapid expansion during the present year due to our efforts during the recent past at purifying the Glycera monomer fraction. Of prime importance in this effort is the fact that one of these components has been determined to lack the distal histidine. Studies of these proteins will include ligation dynamics, equilibrium ligation studies and these will be related to the spectroscopic characterization of the heme pocket in conjunction with sequence and structural studies carried out elsewhere. Conclusions drawn from these studies will have extensive impact upon theories of the particular role which distal residues play in both cooperative and non-cooperative ligand binding processes. These will be true structure-function correlations which may define specific factors which govern the primary process (ligand binding) of non-covalent heme proteins. The results of this research will impact the design of synthetic heme enzymes and tailored heme proteins.