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: 3229733
• 负责人: James D. Satterlee
• 金额: $ 10.87万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-09-30 至 1990-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3229733
• 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.

本提案中所述的工作是我们继续开展的工作的延伸。 努力描述和比较两个独特的血红素活性位点的动力学 proteins. 其中之一是细胞色素c过氧化物酶，一种亚铁血红素酶 在詹姆斯·厄曼博士的实验室里从面包酵母中分离出来。 的 另一种蛋白质实际上是一组蛋白质， 在我自己的实验室中分离和纯化， 二鳃单囊藻的“Moglobin血红蛋白”级分。 细胞色素c 过氧化物酶（CcP）已经通过不太精确的光谱方法进行了研究， 但我们的初步研究表明质子核磁共振 光谱学可以有效地用于确定所发挥的作用， 特定的群体，无论是对蛋白质和血红素组，这影响 酶的反应性（见下文，也见重印本和预印本）。 我们计划继续我们的动力学研究与核磁共振采用饱和 转移和Redfield脉冲序列实验。 这将导致 超精细移动质子的进一步分配，血红素结合的分析 动力学，阐明超精细移动质子的不稳定性， 外源分子之间通讯程度的定量 以及作为活性部位的组成部分的单个原子。 我们的工作 在第二组蛋白质中， 今年由于我们在最近的过去在净化的努力， 甘油单体级分。 在这项努力中，最重要的是， 其中一个组件被确定缺少远端 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 血红素口袋的表征结合序列和 在其他地方进行的结构研究。 由此得出的结论 研究将对特定角色的理论产生广泛的影响 末端残基在合作和非合作配体中起作用 绑定过程。 这些将是真正的结构-功能相关性 其可以定义控制主要过程的特定因素（配体 结合）的非共价血红素蛋白。 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。