Control of Heme Enzyme Activity

血红素酶活性的控制

• 批准号: 9513047
• 负责人: James Erman
• 金额: $ 24万
• 依托单位: Northern Illinois University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9513047&HistoricalAwards=false
• 关键词: Control; Heme; Enzyme; Activity

9513047 Erman The long-term goal of this research is to elucidate the structural features which control heme protein reactivity and determine heme protein function. The research will compare proteins from two different functional classes: (1) the heme enzymes and (2) the oxygen transport/storage proteins. Of particular interest is the chemical reactivity of the Fe(III) state of heme proteins. The properties of CcP will be compared with those of the Fe(III) state of two oxygen transport/storage proteins, mammalian metmyoglobin (MetMb) and Glycera dibranchiata methemoglobin (metGHb) in an effort to understand the structural features which are responsible for the reactivity differences in these two classes of heme proteins. The first aspect of Fe(III) heme protein reactivity to be investigated is the reaction which characterizes the peroxidases, the reaction between the Fe(III) protein and H2O2 to form an oxidized enzyme intermediate called Compound I. Initial studies, using site-directed mutagenesis to alter protein structure, indicate that two amino acid residues in CcP are critical in the formation of Compound I, the distal histidine His-52 and a distal arginine (Arg-48). Arg-48 facilitates oxygen-oxygen bond cleavage while His-52 promotes peroxide binding to the heme iron. Elucidation of the differences in the properties of the distal histidine in the peroxidases and the oxygen transport/storage heme proteins is one of the specific objectives of this proposal. A key question concerning the reactivity of the Fe(III) state of heme proteins is access of small reactants and ligands to the distal heme pocket. Active-site access will be probed by investigating the rates of binding of small ligands such as fluoride, cyanide, and nitric oxide to the iron in CcP, metMb, metGHb and their mutants. Discrimination between the neutral and charged forms of the ligands will be determined. Steric constraints, acid-base behavior, and the polarity of the heme pocket will be investigated through site-directed mutagenesis. The studies outlined in this proposal will provide a more sophisticated level of understanding of heme protein reactivity and function. It will provide a basis for rational protein design. The proposal includes plans to convert metGHb into an efficient peroxidase and more importantly, to convert CcP into a versatile hydroxylase, utilizing H2O2 rather than O2. %%% Heme proteins perform many important functions in living organisms including the transport of oxygen to its site of utilization and the protection of the organism from reactive oxygen species. (Reactive oxygen species such as superoxide, hydrogen peroxide, and the hydroxyl radical are thought to accelerate the aging process.) The goal of the research proposed in this project is to determine how the structure of various heme proteins govern their biological function. Two heme proteins, with different functions, will be extensively investigated by systematic manipulation of their structures. Observation of the biological reactivity of the altered proteins will allow correlations to be made concerning structure and function. The studies will provide a sophisticated level of understanding of heme protein reactivity and function. These studies will also provide a basis for the rational design of heme proteins to carry out processes which may have important commercial applications. ***

9513047这项研究的长期目标是阐明控制血红素蛋白反应性并确定血红素蛋白功能的结构特征。该研究将比较来自两个不同功能类别的蛋白质：（1）血红素酶和（2）氧运输/储存蛋白。特别值得关注的是Fe（III）血红素蛋白的化学反应性。 CCP的特性将与Fe（III）的两种氧运输/储存蛋白，哺乳动物Metmyoglobin（METMB）和糖二伯氏抗甲性脱脂蛋白（METGHB）的特性进行比较，以了解这些蛋白质两类的反应性差异。 Fe（III）血红素蛋白反应性的第一方面是表征过氧化物酶，Fe（III）蛋白和H2O2之间的反应，形成一个氧化酶的酶中间研究，使用位置定向的诱变对蛋白质的结构改变了蛋白质，表明CCP在CCP中的两个氨基酸残基在CCP中的两种氨基酸均具有关键的形成，该酶是在CCP中进行了定位的诱变。远端精氨酸（ARG-48）。 ARG-48促进氧氧键裂解，而HIS-52促进过氧化氧化物与血红素铁的结合。 阐明过氧化物酶远端组氨酸和氧运输/储存血红素蛋白的差异是该提案的特定目标之一。 有关Fe（III）状态血红素蛋白反应性的关键问题是将小反应物和配体进入远端血红素袋。通过研究小体配体的结合速率，例如氟化物，氰化物和一氧化氮与CCP，METMB，METGHB及其突变体中的铁与铁的结合速率来探测活跃的位置。将确定配体的中性和带电形式之间的区分。空间约束，酸碱行为和血红素口袋的极性将通过定位的诱变进行研究。该提案中概述的研究将提供对血红素蛋白反应性和功能的更复杂的理解水平。它将为理性蛋白质设计提供基础。该提案包括将MetGHB转换为有效的过氧化物酶，更重要的是，使用H2O2而不是O2将CCP转换为多功能羟化酶。 %% %%血红素蛋白在活生物体中发挥许多重要功能，包括将氧气运输到其利用部位以及保护生物免受活性氧的保护。 （反应性氧种类，例如超氧化物，过氧化氢和羟​​基自由基被认为可以加速衰老过程。）该项目中提出的研究的目标是确定各种血红素蛋白的结构如何控制其生物学功能。两种具有不同功能的血红素蛋白将通过系统地操纵其结构进行广泛研究。观察改变的蛋白质的生物反应性将允许有关结构和功能的相关性。这些研究将提供对血红素蛋白反应性和功能的复杂理解水平。这些研究还将为血红素蛋白的合理设计提供基础，以执行可能具有重要商业应用的过程。 ***