CAREER: Structural and Mechanistic Studies of Phosphoryl Group Transfer Reactions

9983447乳酸磷酸化核苷酸在许多生物过程中起着关键作用，如信号转导、能量储存和新陈代谢，以及遗传密码的维持，仅举几例。催化合成这些重要分子的酶被称为核苷激酶(NKs)或核苷一磷酸酶(NMPKs)，这取决于它们是否催化第一或第二磷酸基加成到核苷上(核苷是核苷酸的非磷酸化形式，可以通过5‘-羟基糖基连接一个、两个或三个磷酸基团)。在这种磷酰化转移反应中，磷酰化供体，通常是三磷酸腺苷(ATP)，将其磷酸化部分转移到受体核苷或核苷酸上。尽管这一反应非常重要，但酶的作用机制仍存在争议。此外，关于负责这一反应的酶缺乏重要信息。这个项目的目的是增加我们在分子水平上对NKs和NMPKs的功能及其催化反应的了解。为此，我们将重点介绍每一类中的一个代表：NK单纯疱疹病毒1型胸苷激酶(HSV1-tk)和NMPK胸苷酸激酶(TMPK)。我们的策略是通过X射线结晶学技术可视化反应路径上发生的各种状态。这意味着能够形成正向和反向(即在两种底物或两种产物都存在的情况下)以及过渡态的稳定的基态蛋白质晶体。这将通过利用底物类似物来捕获处于所需状态的酶，并通过使用无机分子氟化铝来模拟转移的磷酰基来实现。由于以下两个主要原因，上述酶被选择用于本研究。第一，有两种类型的TMPK，其催化残基来自酶的不同区域。这两种类型的比较将有助于揭示这些残基的确切功能，并有助于区分磷酸基转移的缔合和解离机制。HSV1-tk具有NK和NMPK活性。了解这种酶是如何实现这一双重功能的是非常重要的，也将有助于揭示其催化机制。

9983447LaviePhosphorylated nucleotides play a critical role in many biological processes, such as signal transduction, energy storage and metabolism, and the maintenance of the genetic code, to name only a few. The enzymes that catalyze the synthesis of these important molecules are called nucleoside kinases (NKs) or nucleoside monophosphate kinases (NMPKs) depending if they catalyze the addition of the first or the second phosphoryl group to a nucleoside (a nucleoside is the unphosphorylated form of a nucleotide that can have one, two, or three phosphoryl groups attached to it through the 5'-hydroxyl sugar moiety). In such a phosphoryl transfer reaction, a phosphoryl donor, usually adenosine triphosphate (ATP) transfers its phosphoryl moiety to an acceptor nucleoside or nucleotide. Despite the great importance of this reaction, the enzymatic mechanism is still disputed. Additionally, significant information is lacking about the enzymes that are responsible for this reaction. It is the aim of this project to increase our understanding at the molecular level about how NKs and NMPKs function and the reaction they catalyze.To this aim, we will focus on a representative from each class: the NK herpes simplex virus type 1 thymidine kinase (HSV1-tk), and the NMPK thymidylate kinase (TMPK). Our strategy is to visualize, through the technique of X-ray crystallography, the various states that occur along the reaction pathway. That means being able to form stable protein crystals of the ground states of both the forward and backward directions (i.e. in the presence of both substrates or both products), and of the transition state. This will be achieved by the utilization of substrate analogs that trap the enzyme in the desired state, and by using the inorganic molecule aluminum fluoride to mimic a transferred phosphoryl group.The above enzymes have been chosen for this study due to the following two main reasons. One, there are two types of TMPKs, with catalytic residues originating from different regions of the enzyme. The comparison between the two types will help to reveal the exact function of these residues, and aid in differentiating between the associative and dissociative mechanisms of phosphoryl transfer. Two, HSV1-tk has both NK and NMPK activity. Understanding how this enzyme achieves this dual function is very important and will also shed light on the catalytic mechanism.