Sulfate Adenylation-Biochemistry & Enzymology

硫酸腺苷酸化-生物化学

• 批准号: 7600570
• 负责人: Thomas S. Leyh
• 金额: $ 52.89万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-02 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7600570
• 关键词: ATP Hydrolysis; Active Sites; Address; Adenylylsulfate kinase; Allosteric Site; Amino Acids; Area; Assimilations; Bacteria; Behavior; Binding; Binding Sites; Biochemistry; Biological; Biology; Breast; Catalytic Domain; Cells; Chemicals; Complex; Coupled; Cysteine; Data; Electrostatics; Endometrial Neoplasms; Endometrium; Entropy; Environment; Enzymatic Biochemistry; Enzymes; Escherichia coli; Estradiol; Estrone sulfotransferase; Event; Exhibits; Fluorescence Resonance Energy Transfer; Funding; Goals; Grant; Growth; Hand; Hemophilia B; Human; Hydrolysis; Immune system; In Vitro; Inorganic Sulfates; Isotopes; Length; Life; Ligand Binding; Link; Malignant Neoplasms; Mammals; Measurement; Metabolic; Metabolism; Modeling; Molecular; Molecular Machines; Movement; Mycobacterium tuberculosis; Optics; Organism; Pathway interactions; Phenylalanine; Plant Roots; Positioning Attribute; Production; Reaction; Resolution; Series; Shapes; Site; Spectrum Analysis; Structural Models; Structure; Sulfate Adenylyltransferase; Sulfur; Sulfur Metabolism Pathway; Surface; System; Techniques; Testing; Time; TimeLine; Travel; Tubular formation; Unspecified or Sulfate Ion Sulfates; Ursidae Family; Work; base; complex R; crosslink; dimer; driving force; fascinate; human disease; monomer; public health relevance; reactive site (enzyme); research study; response; simulation; sulfotransferase

DESCRIPTION (provided by applicant): The metabolism of sulfur presents a complex and fascinating molecular network whose activities impact many areas of biology, including human disease. We now know that at least four of the enzymes involved in sulfur assimilation in bacteria organize into a multifunctional complex from which new catalytic function (ATP hydrolysis) emerges. Remarkably, this hydrolysis is kinetically and energetically linked, via conformational changes, to turnover of the first enzyme in the cysteine biosynthetic pathway - ATP sulfurylase. We intend to explore the mechanism of this linkage and to determine the composition and organization of the cysteine-metabolome both in vitro and in the environment of a living cell. We have discovered that in Type III sulfate activating complexes (SACs), activated sulfate (APS) travels between the active sites that produce and consume it along a deep 75 E-long groove that opens and closes in response to the position of APS. Using FRET, we will test the hypothesis that the channel closes to form a tubular structure during APS transit. Combining pre-steady state and FRET measurements, we will construct a timeline that interdigitates the events that occur in the catalytic cycle with changes in distance along the length of the channel. Using Brownian Dynamics we will advance a cutting-edge model of how changes in the shape and electrostatics of this remarkable molecular machine are coupled to the movement of the APS within it. Transfer of the sulfuryl- moiety (SO3) from activated sulfate to biological recipients is used widely by the cell to regulate metabolism. Sulfotransferases, which catalyze these transfers, are subject to allosteric substrate inhibition that is not well understood primarily because extensive structural and function work has not identified an allosteric binding pocket. The human estrogen sulfotransferase (EST) exhibits a presteady-state product burst that corresponds to precisely one-half of the active sites in the dimer. If EST is a half-site reactive enzyme, the non-catalytic active site might well function as the allosteric site of inhibition. We will test this hypothesis using the human EST, an enzyme whose activity is tightly and causally linked to cancer in the breast and endometrium. PUBLIC HEALTH RELEVANCE Transfer of the sulfuryl-group (SO3) from activated sulfate to various metabolic recipients is used widely by the cell to regulate function. Sulfotransferases, which catalyze these reactions, are themselves regulated by allosteric substrate inhibition, the molecular mechanism of which is unknown despite considerable effort to the contrary. We believe we now understand this mechanism, and will prove our mechanistic hypotheses in the upcoming grant period. The actions of these enzymes are tightly, causally linked to numerous human disease conditions, including: hemophilia B, compromised immune systems, androgyny, and breast and endometrial tumors. In a second Aim, we will explore a rare complex found in M. tuberculosis. This complex, which we discovered, is not present in mammals and therefore holds the promise of species-specific inhibition. This cysteine metabolome is comprise of at least four of the six enzymes in the cysteine biosynthetic pathway, and exhibits remarkable catalytic synergies. We will characterize this complex in detail, and for the first time.

描述（由申请人提供）：硫的代谢提出了一个复杂而引人入胜的分子网络，其活动影响了许多生物学领域，包括人类疾病。我们现在知道，在细菌中至少有四种涉及硫同化的酶，它们组织成多功能复合物，从中出现了新的催化功能（ATP水解）。值得注意的是，这种水解通过构象变化在动力学和能量上是与半胱氨酸生物合成途径 - ATP硫酶中第一种酶的营业率相连的。我们打算探索这种联系的机制，并在体外和活细胞的环境中确定半胱氨酸 - 替代体的组成和组织。我们发现，在III型硫酸盐激活复合物（SAC）中，激活的硫酸盐（APS）在产生和消耗的活性位点之间行驶，沿着75个E-75 E-E-E-E-E-E-E-E-E-long凹槽消耗，该凹槽响应于AP的位置。使用FRET，我们将检验以下假设：通道在APS传输过程中关闭以形成管状结构。结合稳态状态和FRET测量值，我们将构建一个时间表，该时间表将催化周期中发生的事件与沿通道长度的距离变化发生变化。使用布朗动力学，我们将推进一个尖端的模型，该模型如何将这种非凡的分子机器的形状和静电变化与其中的AP运动耦合。细胞广泛使用硫酸盐（SO3）从活化的硫酸盐到生物学接受者的转移来调节代谢。催化这些转移的硫代转移酶受到变构底物的抑制作用，这主要是因为广泛的结构和功能工作尚未鉴定出变构结合口袋。人类雌激素硫代转移酶（EST）表现出一种质状态的产物爆发，对应于二聚体中活性位点的一半。如果EST是半位点反应酶，则非催化活性位点可能充当抑制的变构位点。我们将使用人类EST（一种酶，其活性与乳腺癌和子宫内膜的癌症紧密相关的酶，我们将使用人类EST进行检验。 硫酸组（SO3）从活化的硫酸盐到各种代谢受体的公共卫生相关性转移被细胞广泛使用以调节功能。促催化这些反应的硫代转移酶本身受到变构底物抑制的调节，尽管相反的努力很大，但其分子机制还是未知的。我们相信我们现在了解这种机制，并将在即将到来的赠款期间证明我们的机械假设。这些酶的作用紧密，因果关系与许多人类疾病疾病有关，包括：血友病B，免疫系统受损，雄孕，乳腺癌和子宫内膜肿瘤。在第二个目标中，我们将探索结核分枝杆菌中发现的罕见复合物。我们发现的这种复合物不存在于哺乳动物中，因此具有特异性抑制的希望。这种半胱氨酸代谢组包括半胱氨酸生物合成途径中六种酶中的至少四种，并表现出显着的催化协同作用。我们将首次详细介绍这个复合体。