CAREER: Structural and Mechanistic Studies of Phosphoryl Group Transfer Reactions

University Of Illinois At Chicago, Chicago IL

Investigators

Abstract

9983447 Lavie Phosphorylated 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.