Investigating the interaction between GTP-cyclohydrolase1 and its feedback regulatory protein

Abstract

Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic amino acid-hydroxylases and all isoforms of nitric oxide-synthase. In the cardiovascular system nitric oxide (NO) elicits potent vasodilatory and antithrombotic effects and maintains endothelial function. Administration of BH4 can improve endothelial function in numerous cardiovascular pathologies, an effect that likely results from increased NO bioavailability and/or local antioxidant effects. GTP cyclohydrolase1 (GCH1) is the rate-limiting enzyme for BH4 biosynthesis. GCH1 activity has been shown to be modified by both levels of phenylalanine and BH4 through allosteric regulation when bound to GCH1-feedback regulatory protein (GFRP). Studies have shown that phenylalanine binds to the complex and activates GCH1, whilst BH4 binding to the complex inhibits GCH1 activity. Our studies aim to investigate the significance of this protein–protein interaction on BH4 bioavailability. We have shown that oral phenylalanine administration (100 mg/kg) in rodents leads to an increase in BH4 production in aortic tissue, suggesting that targeting the GCH1–GFRP complex may provide a means to regulate vascular BH4 bioavailability. Using recombinantly expressed human proteins; the nature of the GCH1–GFRP interaction has been investigated using biophysical techniques. In addition the activity of purified recombinant enzymes has been measured by two methods. Initial results suggest the importance of the first 42 amino acids of the N-terminus of GCH1, and its potential role in mediating the activity of the enzyme and its interaction with GFRP. A greater understanding of GCH1–GFRP protein–protein interactions and how these translate to BH4 bioavailability may help identify novel therapies for a range of cardiovascular diseases.