THERMODYNAMIC AND KINETIC STUDIES OF PROTEIN STRUCTURE AND ENZYMIC MECHANISMS

蛋白质结构和酶机制的热力学和动力学研究

• 批准号: 6161904
• 负责人: P MC PHIE
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6161904
• 关键词: amphotericin B; chemical kinetics; circular dichroism; conformation; enzyme activity; enzyme mechanism; high performance liquid chromatography; histidine; protein folding; protein sequence; protein structure; sulfotransferase; thermodynamics; tryptophan synthase

This laboratory is interested in the relationship between protein sequence, structure and the mechanisms of protein folding and enzymic reactions. (i) Histidine Rich Proteins The malaria parasite, Plasmodium falciparum, produces large amounts of several histidine rich proteins of unknown structure. Their high concentrations of histidine distort their circular dichroic spectra, so that, unlike those of normal proteins, they cannot be interpreted in terms of secondary structures. We have reinvestigated the behaviour of poly L-histidine as a function of pH and solvent additives, and established a set of reference spectra under defined conditions, which can be used in analysis of the structures of these unusual proteins. (ii) Structure of Tryptophan Synthase (with S. Ashraf Ahmed and Edith Wilson Miles, LBP) We are investigating the conformational states of the tryptophan synthase alpha2-beta2 complex and of the separate alpha and beta subunits, using site directed mutagenesis. During catalysis, the enzyme changes from an open to a closed form, excluding solvent from the active site and facilitating chemical reactions. Analysis of a series of double mutants show that the more active closed form is stabilised by electrostatic interactions in the beta subunit. (iii) Structure of Mammalian Sulphotransferases. (with D. Marshall and W.B. Jakoby, LBM) The enzymatic activity of a phenol sulphotransferase, from rat liver, has been shown to be regulated by reversible oxidation/reduction of a conserved cysteine residue by physiological concentrations of glutathione. The two forms of the protein show differing complex patterns of inhibition by substrates and products, which indicate that the two active sites in the dimeric enzyme, although identical in sequence have very different chemical properties (half of sites reactivity).

该实验室对蛋白质之间的关系感兴趣 蛋白质折叠和酶促的序列、结构和机制 反应。 (i) 富含组氨酸的蛋白质 疟原虫， 恶性疟原虫，产生大量富含组氨酸的物质 结构未知的蛋白质。它们的组氨酸浓度很高 扭曲它们的圆二向色光谱，因此，与正常的光谱不同 蛋白质，它们不能用二级结构来解释。 我们重新研究了聚 L-组氨酸作为函数的行为 pH值和溶剂添加剂的影响，并建立了一套参考光谱 在定义的条件下，可用于分析 这些不寻常的蛋白质的结构。 (ii) 色氨酸的结构 Synthase（与 S. Ashraf Ahmed 和 Edith Wilson Miles，LBP） 研究色氨酸合酶的构象状态 α2-β2复合物和单独的α和β亚基，使用 定点诱变。在催化过程中，酶从 开放至封闭形式，排除活性位点中的溶剂，并且 促进化学反应。一系列双突变体的分析 表明更活跃的闭合形式通过静电稳定 β亚基中的相互作用。 (iii) 哺乳动物的结构 磺基转移酶。 （与 D. Marshall 和 W.B. Jakoby，LBM） 大鼠肝脏中苯酚磺基转移酶的酶活性 已被证明是通过可逆氧化/还原来调节的 生理浓度的保守半胱氨酸残基 谷胱甘肽。蛋白质的两种形式表现出不同的复杂性 底物和产物的抑制模式，这表明 二聚酶中的两个活性位点，尽管在 序列具有非常不同的化学性质（一半位点 反应性）。