Tetrahydrobiopterin (BH4) serves as an essential cofactor for the enzymes that produce nitric oxide, serotonin, dopamine, epinephrine, and norepinephrine. The activities of these enzymes are regulated by the availability of BH4 in the cells. Our long-range objectives of this research are to understand the mechanism that regulates GTP cyclohydrolase I, the first and rate-limiting enzyme in the biosynthetic pathway for BH4. The activity of GTP cyclohydrolase I is controlled by a regulator protein. This key protein is called GTP cyclohydrolase I feedback regulator protein (GFRP), which inhibits the enzyme activity in the presence of BH4 and stimulates it in the presence of phenylalanine through ligand-induced complex formation. During the past funded period we have demonstrated that in both the inhibitory and stimulatory complexes two molecules of GFRP, a pentamer, are physically associated with one molecule of GTP cyclohydrolase I, a decamer. Approximately one molecule of each ligand was found to bind to the respective protein complexes per subunit of the two proteins. Our current working hypothesis is that the catalytic function of GTP cyclohydrolase I is determined by two different conformational states of the protein complexes that are formed by the binding of respective effectors. To test this hypothesis and obtain further information on the regulatory mechanisms of this system, we now propose the following three aims.
AIM 1 : Define the relationship among enzyme activity, ligand binding, and GFRP binding.
AIM II : Determine the three- dimensional structures of GTP cyclohydrolase I, GFRP, the inhibitory complex, and the stimulatory complex, and characterize the conformational transition between these structures.
AIM III : Define the functional role of phosphorylation in the GTP cyclohydrolase I-GFRP system and determine the phosphorylation sites of GTP cyclohydrolase I and GFRP. Detailed understanding of the regulatory mechanisms of the GTP cyclohydrolase I/GFRP system will greatly enhance knowledge of metabolism of catecholamines, serotonin, and nitric oxide as well as BH4. The knowledge will provide insight into the altered metabolism of BH4 reported in phenylketonuria, Parkinson's disease, familial dystonia, and several vascular diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK051257-06
Application #
6688934
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Sechi, Salvatore
Project Start
1997-09-01
Project End
2005-11-30
Budget Start
2003-12-01
Budget End
2005-11-30
Support Year
6
Fiscal Year
2004
Total Cost
$212,028
Indirect Cost
Name
University of Pittsburgh
Department
Surgery
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Chen, Wei; Bacanamwo, Methode; Harrison, David G (2008) Activation of p300 histone acetyltransferase activity is an early endothelial response to laminar shear stress and is essential for stimulation of endothelial nitric-oxide synthase mRNA transcription. J Biol Chem 283:16293-8
Widder, Julian D; Chen, Wei; Li, Li et al. (2007) Regulation of tetrahydrobiopterin biosynthesis by shear stress. Circ Res 101:830-8
Maita, Nobuo; Hatakeyama, Kazuyuki; Okada, Kengo et al. (2004) Structural basis of biopterin-induced inhibition of GTP cyclohydrolase I by GFRP, its feedback regulatory protein. J Biol Chem 279:51534-40
Maita, Nobuo; Okada, Kengo; Hatakeyama, Kazuyuki et al. (2002) Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP. Proc Natl Acad Sci U S A 99:1212-7
Collins, J L; Vodovotz, Y; Yoneyama, T et al. (2001) Catecholamines decrease nitric oxide production by cytokine-stimulated hepatocytes. Surgery 130:256-64
Yoneyama, T; Wilson, L M; Hatakeyama, K (2001) GTP cyclohydrolase I feedback regulatory protein-dependent and -independent inhibitors of GTP cyclohydrolase I. Arch Biochem Biophys 388:67-73
Yoneyama, T; Hatakeyama, K (2001) Ligand binding to the inhibitory and stimulatory GTP cyclohydrolase I/GTP cyclohydrolase I feedback regulatory protein complexes. Protein Sci 10:871-8
Maita, N; Okada, K; Hirotsu, S et al. (2001) Preparation and crystallization of the stimulatory and inhibitory complexes of GTP cyclohydrolase I and its feedback regulatory protein GFRP. Acta Crystallogr D Biol Crystallogr 57:1153-6
Geller, D A; Di Silvio, M; Billiar, T R et al. (2000) GTP cyclohydrolase I is coinduced in hepatocytes stimulated to produce nitric oxide. Biochem Biophys Res Commun 276:633-41
Yoneyama, T; Hatakeyama, K (1998) Decameric GTP cyclohydrolase I forms complexes with two pentameric GTP cyclohydrolase I feedback regulatory proteins in the presence of phenylalanine or of a combination of tetrahydrobiopterin and GTP. J Biol Chem 273:20102-8