The overarching goal of this proposal is to improve upon the current assays that monitor S- Adenosylmethionine-dependent enzymes and ATP-dependent enzymes. ATP and S-adenosylmethionine (AdoMet) are the two most common substrates in all living organisms. Having recently shown that the bacterial enzyme Methylthioadenosine Nucleosidase (MTAN) can readily bind the products (ADP and S-adenosylhomocysteine) derived from enzymatic reactions using either of these substrates, we propose to engineer new variants of this enzyme to improve affinity to these compounds. These variants will form the basis for mix-and-read, continuous assays that can be coupled to any ATP or AdoMet-utilizing enzyme. The first portion of the study will make MTAN variants and test them for binding to either AdoMet or AdoHcy to assess affinity. In the second aim, other MTAN variants will be made and affinity to ATP, ADP, and AMP will be tested. It is expected that results obtained from this study will product two marketable products for in vitro enzymatic testing: one for AdoMet-dependent enzymes and one for ATP-dependent enzymes.
Public Health Relevance Statement: Project Narrative Drug discovery efforts that lead to new treatments for Cancer, Heart Disease, Infectious Diseases, and countless other human ailments focus on identifying and optimizing compounds that inhibit enzyme function. This research aims to develop more sensitive and more rapid tools for identifying and testing potential drugs.
Project Terms: Affinity; Amino Acids; Binding; Biochemical Reaction; Biological Assay; cancer therapy; Cells; Communicable Diseases; Competitive Binding; Complex; cost; Coupled; Coupling; Crystallization; design; Detection; drug discovery; Dyes; Engineering; Enzymes; Exhibits; experimental study; Fluorescence Polarization; Fluorescent Probes; Goals; Heart Diseases; Helicobacter pylori; Human; improved; In Vitro; inorganic phosphate; Laboratories; Lead; Methods; Methyltransferase; Monitor; Mutate; Nucleoside Hydrolases; Organism; Paper; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phosphotransferases; Production; real time monitoring; Reporting; Reproducibility; Research; Research Project Grants; Roentgen Rays; S-Adenosylhomocysteine; S-Adenosylmethionine; Signal Transduction; Speed; Structure; Testing; thioether; Time; tool; Variant
