Determination of enzyme isotope effects by tandem ESI-Q/TOF mass spectrometry

通过串联 ESI-Q/TOF 质谱测定酶同位素效应

• 批准号: 7191481
• 负责人: MICHAEL E. HARRIS
• 金额: $ 11.59万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7191481
• 关键词: Acids; Active Sites; Acute; Affect; Animal Viruses; Biochemical; Biology; Catalysis; Catalytic RNA; Chemistry; Chronic; Class; Cleaved cell; Cytosine; DNA; Development; Electrospray Ionization; Environment; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Equilibrium; Face; Future; Growth; Growth and Development function; Hepatitis Delta Virus; Human; Hydrolysis; Ions; Isotopes; Kinetics; Left; Life; Light; Liver diseases; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Metals; Methods; Modeling; Modification; Nucleic Acids; Nucleosides; Nucleotides; Object Attachment; Oligonucleotides; Oxygen; Pharmaceutical Preparations; Phosphorus; Phosphotransferases; Property; Proteins; Publishing; RNA; Rate; Reaction; Reagent; Ribonucleases; Ribose; Secondary to; Series; Site; Spectrometry; Spectrometry, Mass, Electrospray Ionization; Staging; Standards of Weights and Measures; Structure; Techniques; Testing; Therapeutic; Time; Viral; Virus; base; chemical reaction; circular RNA; design; endonuclease; enzyme mechanism; inhibitor/antagonist; inorganic phosphate; interest; ionization; novel; nucleobase; pathogen; protonation; research study; stable isotope; therapeutic target

DESCRIPTION (provided by applicant): When stable isotopes are substituted for atoms undergoing a chemical reaction, it changes the rates and equilibria of the reactions in which the substituted atom is involved. By analyzing these isotope effects (lEs) for enzyme-catalyzed reactions one can elucidate details of enzyme mechanism and provide key information about active site interactions. This information is essential for understanding how the enzymes that are essential for human life function. Importantly, this information can be used to design specific inhibitors of enzyme function that can be used to stop the growth of cancer and viruses. However, due to technical limitations IE analysis is not widely applied to enzymes acting on DMA and RNA. These enzymes are of intense interest because they are fundamental to biology and are important targets for therapeutics, yet gaining direct information on their mechanism has been difficult. Defining the mechanism of nucleic acid phosphoryl transfer enzymes by competitive IE analyses faces two substantial challenges- first, sufficient amounts of DNA or RNA with site-specific isotopic enrichment must be synthesized, and second, the isotopic composition of the product must be precisely determined by mass spectrometry (MS). Our recent results on nucleophile clearly demonstrate the feasibility of using a new analytical technique, whole molecule electrospray ionization MS (ESI-MS), for measuring lEs on reactions of RNA oligonucleotides. Additionally, we have observed that advances in tandem quadropol / time of flight (Q/TOF) ESI-MS now permit analysis of very small amounts of material with increased precision necessary for detailed IE analysis of enzyme mechanism. In this project we will develop Q/TOF ESI-MS together with standard nucleoside modification chemistry using inexpensive and commercially available reagents to permit the lEs on each of the oxygens of a single, specific scissile phosphate in an oligonucleotide substrate. Such information will set the stage for the future design of active site based inhibitors as anti-proliferative (anri- cancer) or anti viral drugs.

描述（由申请人提供）：当稳定同位素取代正在进行化学反应的原子时，它会改变取代原子所涉及的反应的速率和平衡。通过分析酶催化反应的这些同位素效应 (IE)，我们可以阐明酶机制的细节，并提供有关活性位点相互作用的关键信息。这些信息对于了解人类生命所必需的酶如何发挥作用至关重要。重要的是，这些信息可用于设计特定的酶功能抑制剂，用于阻止癌症和病毒的生长。然而，由于技术限制，IE 分析并未广泛应用于作用于 DMA 和 RNA 的酶。这些酶引起了人们的浓厚兴趣，因为它们是生物学的基础，也是治疗的重要靶点，但获得有关其机制的直接信息却很困难。通过竞争性 IE 分析来定义核酸磷酰转移酶的机制面临着两个重大挑战：首先，必须合成足够量的具有位点特异性同位素富集的 DNA 或 RNA，其次，必须通过质谱 (MS) 精确测定产物的同位素组成。我们最近关于亲核试剂的结果清楚地证明了使用新的分析技术全分子电喷雾电离 MS (ESI-MS) 来测量 RNA 寡核苷酸反应的 IE 的可行性。此外，我们还观察到，串联四极杆/飞行时间 (Q/TOF) ESI-MS 的进步现在可以分析极少量的材料，同时提高了酶机制详细 IE 分析所需的精度。在该项目中，我们将开发 Q/TOF ESI-MS 以及标准核苷修饰化学，使用廉价且市售的试剂，以允许寡核苷酸底物中单个特定裂解磷酸盐的每个氧上存在 IE。这些信息将为未来设计基于活性位点的抑制剂作为抗增殖（抗癌）或抗病毒药物奠定基础。