CAREER: Structural and Mechanistic Studies of Phosphoryl Group Transfer Reactions

9983447 lavi磷酸化核苷酸在许多生物过程中起着至关重要的作用，例如信号转导，能量储存和代谢，以及遗传密码的维持，仅举几例。催化这些重要分子合成的酶被称为核苷激酶（NKs）或核苷单磷酸激酶（NMPKs），这取决于它们是催化第一个还是第二个磷酸化基团到核苷上（核苷是核苷酸的未磷酸化形式，可以通过5'-羟基糖部分连接一个、两个或三个磷酸化基团）。在这种磷酸基转移反应中，磷酸基供体，通常是三磷酸腺苷（ATP）将其磷酸基部分转移到受体核苷或核苷酸上。尽管这一反应非常重要，但其酶促机制仍存在争议。此外，关于负责该反应的酶的重要信息缺乏。这个项目的目的是增加我们在分子水平上对NKs和NMPKs如何起作用以及它们催化的反应的理解。为此，我们将重点介绍每一类的代表：NK单纯疱疹病毒1型胸苷激酶（HSV1-tk）和NMPK胸苷激酶（TMPK）。我们的策略是可视化，通过x射线晶体学技术，在反应过程中发生的各种状态。这意味着能够形成稳定的正反向基态（即在两种底物或两种产物存在的情况下）和过渡态的蛋白质晶体。这将通过利用底物类似物使酶处于所需状态，并通过使用无机分子氟化铝来模拟转移的磷酰基来实现。选择上述酶进行本研究主要有以下两个原因。首先，有两种类型的tmpk，催化残基来自酶的不同区域。两种类型之间的比较将有助于揭示这些残基的确切功能，并有助于区分磷酰转移的结合和解离机制。第二，HSV1-tk同时具有NK和NMPK活性。了解这种酶如何实现这种双重功能是非常重要的，也将揭示催化机制。

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.