STUDIES OF THE DYNAMICS OF HEME PROTEIN ACTIVE SITES

血红素蛋白活性位点的动力学研究

• 批准号: 3229734
• 负责人: James D. Satterlee
• 金额: $ 9.6万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1991-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3229734
• 关键词: Annelida; Raman spectrometry; aminoacid; chemical binding; chemical reaction; chemical structure function; cytochrome c; electrofocusing; electron spin resonance spectroscopy; enzyme mechanism; enzyme structure; erythrocytes; heme; hemoglobin; hemoprotein structure; high performance liquid chromatography; histidine; infrared spectrometry; ion exchange chromatography; ligands; mathematical model; molecular site; myoglobin; nuclear magnetic resonance spectroscopy; peroxidases; protein engineering; saltwater environment; stereochemistry; ultraviolet spectrometry

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.

本提案中描述的工作是我们持续的延伸 努力描述和比较两种独特血红素的活性位点动态 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 在 James Erman 博士的实验室中从面包酵母中分离出来。 这 其他蛋白质实际上是一组被单独分离的蛋白质 在我自己的实验室中分离和纯化，其中包括所谓的 Glycera Dibranchiata 的“单体血红蛋白”部分。 细胞色素c 过氧化物酶 (CcP) 已通过不太精确的光谱方法进行研究， 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于识别所起的作用 影响蛋白质和血红素基团的特定基团 酶的反应性（见下文，另见重印本和预印本）。 我们计划继续利用饱和核磁共振进行动力学研究 转移和雷德菲尔德脉冲序列实验。 这些将导致 超精细位移质子的进一步分配，血红素结合分析 动力学，阐明超精细移位质子的不稳定性和 外在分子之间通讯程度的定量 以及作为活性位点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扩张的边缘 今年由于我们最近在净化环境方面所做的努力 甘油单体部分。 在这项工作中最重要的是以下事实 这些组件之一已被确定缺乏远端 组氨酸。 这些蛋白质的研究将包括连接动力学， 平衡连接研究，这些将与光谱相关 结合序列和血红素口袋的特征 其他地方进行的结构研究。 从这些得出的结论 研究将对特定作用的理论产生广泛的影响 哪些远端残基在合作配体和非合作配体中都起作用 绑定过程。 这些将是真正的结构-功能相关性 它可以定义控制初级过程的特定因素（配体 非共价血红素蛋白的结合）。 这项研究的结果将 影响合成血红素酶和定制血红素蛋白的设计。