Structure-Function of Metalloenzymes

金属酶的结构-功能

• 批准号: 7659889
• 负责人: CAROL A FIERKE
• 金额: $ 4.25万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7659889
• 关键词: Acetylation; Acetylglucosamine; Acids; Active Sites; Affect; Affinity; Alkylation; Amino Acids; Anti-Bacterial Agents; Antibiotics; Antibodies; Antineoplastic Agents; Binding; Biochemical; Biological; Biological Assay; Biological Process; Categories; Cell Differentiation process; Class; Clinical Trials; Commit; Cysteine; Cystic Fibrosis; Data; Deacetylase; Deacetylation; Detection; Development; Differentiation Inhibitor; Dimethylallyltranstransferase; Dissociation; Electronics; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Gene Expression; Gene Expression Regulation; Goals; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Histone Deacetylase; Histones; Human; Human Genome; Hydrolysis; Immunoprecipitation; Ions; Iron; Isotopes; Kinetics; Length; Ligands; Lipid A; Lysine; Measurement; Measures; Metals; Methods; Methyltransferase; Modification; Mutagenesis; N-acetylglucosamine deacetylase; Nature; Organism; Pathway interactions; Peptide Library; Post-Translational Protein Processing; Property; Protein Acetylation; Proteins; Proteome; Proteomics; Reaction; Regulation; Research; Research Personnel; Signal Transduction Pathway; Specificity; Spectrum Analysis; Structure; Substrate Specificity; Sulfur; Techniques; Variant; Zinc; analog; base; combinatorial; design; in vivo; inhibitor/antagonist; metalloenzyme; molecular recognition; novel; pathogen; prenylation; programs; protein farnesyltransferase; protein geranylgeranyltransferase; research study; tissue/cell culture

The goals of this research are to better understand the mechanism, specificity and biological function of several medically important and unique classes of zinc and/or iron metalloenzymes. (1) Protein farnesyltransferase and protein geranylgeranyltransferaseI catalyze the prenylation of many proteins in important signal transduction pathways. We propose to investigate the structure of the catalytic transition state by measuring kinetic isotope effects and the determinants of substrate specificity and product dissociation by mutagenesis. (2) Proteins modified by the prenylation pathwayswill be identified by first assaying the affinity and catalytic activity of libraries of peptides derived from the human genome and then verified as prenylation targets in vivo using modified prenyldiphosphate substrates and antibody detection. Furthermore, immunoprecipitation and proteomic analysis will be used to interrogate native expression of prenylated proteins. Identification of substrates of this pathway is an important step toward understanding the biological functions of these modifications and the downstreamtargets of the chemotherapeutic inhibitors of this pathwaythat are currently in clinical trials. (3) The enzyme UDP-3-0-(R-3-hydroxymyristoyl)-N- acetylglucosamine deacetylase (LpxC) catalyzesthe committed step in the synthesis of Lipid A in gram negative bacteria, making LpxC an antibacterial target. We propose to characterize the catalytic mechanism and metal specificity (Fe or Zn) using structure variation together with kinetic and thermodynamicstudies. These data will facilitate the design of potent LpxC inhibitors as novel antibiotics especially especially against those organisms associated with cystic fibrosis and some of the potential bioterror agents listed as MlAID category A and B priority pathogens. (4) Histone deacetylases catalyze the deacetylation of acetylated lysine residues involved in the regulation of gene expression and cell differentiation; inhibitors of HDACs are currently in clinical trials as anticancer drugs. We proposeto investigate the identity of the catalytic metal in vivo; the catalytic mechanism; and the substrate specificity, using both known substrates and proteomic approaches. Altering the active site metal may be an important regulatory mechanism of metal-dependent deacetylases. The information gained from these experiments will aid our understanding of the biological function of these important post-translational modifications and enhance the development of novel inhibitors.

这项研究的目标是更好地了解 几种具有医学重要和独特的锌和/或铁金属酶的类别。 （1）蛋白质 Farnesylsylansferseas和蛋白质的蛋白质天烷基凝血酶转移酶催化了许多蛋白质在 重要的信号转导途径。我们建议研究催化过渡的结构 通过测量动力学同位素效应和底物特异性和产物的决定因素来表述 通过诱变解离。 （2）通过先前化途径修饰的蛋白质将通过第一鉴定 分析源自人基因组的肽库的亲和力和催化活性，然后 使用改良的前二磷酸底物和抗体检测为体内验证。 此外，免疫沉淀和蛋白质组学分析将用于询问天然表达 蛋白质蛋白质。识别该途径的底物是迈向理解的重要一步 这些修饰的生物学功能和化学治疗抑​​制剂的下游量 该途径目前正在临床试验中。 （3）酶UDP-3-0-（R-3-Hydroxymyristoyl）-n- 乙酰葡萄糖脱乙酰基酶（LPXC）catalyzesthe在脂质a的合成中catalyzesthe以克的合成 阴性细菌，使LPXC成为抗菌靶标。我们建议表征催化机制 以及金属特异性（Fe或Zn），使用结构变化以及动力学和热力学研究。 这些数据将促进有效的LPXC抑制剂作为新型抗生素的设计，尤其是针对 那些与囊性纤维化相关的生物以及一些列为Mlaid的潜在生物疗法 A类优先病原体。 （4）组蛋白脱乙酰基酶催化乙酰化的脱乙酰基化 参与基因表达和细胞分化调节的赖氨酸残基； HDAC的抑制剂是 目前正在临床试验中作为抗癌药物。我们建议研究催化金属在 体内催化机制；以及使用已知底物和蛋白质组学的底物特异性 方法。改变活性位点金属可能是金属依赖性的重要调节机制 脱乙酰基酶。从这些实验中获得的信息将有助于我们对生物学的理解 这些重要的翻译后修饰的功能并增强了新型抑制剂的发展。