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

说明（由申请人提供）：当稳定同位素取代进行化学反应的原子时，它会改变取代原子所参与的反应的速率和平衡。通过分析酶催化反应的同位素效应，人们可以阐明酶反应机理的细节，并提供有关活性位点相互作用的关键信息。这些信息对于了解对人类生命至关重要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酶功能的特异性抑制剂，可用于阻止癌症和病毒的生长。然而，由于技术上的限制，IE分析没有广泛应用于作用于DMA和RNA的酶。这些酶引起了人们的强烈兴趣，因为它们是生物学的基础，也是治疗的重要靶点，但很难获得有关其机制的直接信息。通过竞争性IE分析确定核酸磷酰基转移酶的机制面临两个重大挑战-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，产物的同位素组成必须通过质谱法（MS）精确测定。我们最近的结果亲核试剂清楚地表明，使用一种新的分析技术，全分子电喷雾电离质谱（ESI-MS），用于测量的RNA寡核苷酸反应的IEs的可行性。此外，我们已经观察到，在串联四极杆/飞行时间（Q/TOF）ESI-MS的进步，现在允许分析非常少量的材料与酶机制的详细IE分析所需的增加的精度。在这个项目中，我们将开发Q/TOF ESI-MS与标准核苷修饰化学一起使用廉价和市售的试剂，以允许在寡核苷酸底物中的单个特异性易断裂磷酸的每个氧上的IEs。这些信息将为将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。

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.