The goal of this project is to use sensitizer-linked-substrates (SLS's) to probe the mechanism and mechanics of electron transfer-mediated catalysis in nitric oxide synthase (NOS). Part I involves the design and synthesis of pterin-based SLS's for NOS; both the natural cofactorthat is endogenously bound by the enzyme will be incorporated, as well as aromatic analogues which will serve as fluorescent probes of the pterin binding pocket. In Part II, using the catalytic oxidase domain of iNOS (iNOSoxy), the binding affinities of the pterin probes will be determined, fluorescence techniques will be used to explore structural and electronic perturbations near the active site during catalysis, x-ray crystallography will be utilized to elucidate the structural effects of incorporation of the probe into the protein, and finally, electron transfer studies will be employed to study the mechanism of NOS catalysis, especially the role of the pterin cofactor. In Part III, we will explore the development of these SLS's for use in high-throughput binding assays for NOS inhibitors. Finally, we will determine the efficacy of these NOS specific probes for studies on NOS in complex mixtures of other proteins. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM074406-01A1
Application #
7054284
Study Section
Special Emphasis Panel (ZRG1-F04A (20))
Program Officer
Fabian, Miles
Project Start
2006-01-01
Project End
2007-12-31
Budget Start
2006-01-01
Budget End
2006-12-31
Support Year
1
Fiscal Year
2006
Total Cost
$48,796
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Glazer, Edith C; Nguyen, Yen Hoang Le; Gray, Harry B et al. (2008) Probing inducible nitric oxide synthase with a pterin-ruthenium(II) sensitizer wire. Angew Chem Int Ed Engl 47:898-901