Regulation of tetrahydrobiopterin synthesis by GTP cyclohydrolase feedback regulatory protein

Nitric oxide synthase (NOS) activity is typically limited by the availability of tetrahydrobiopterin (BH4), a required NOS cofactor. GTP cyclohydrolase I (GTPCH) is the first and rate-determining enzyme in the de novo pathway for BH4 biosynthesis. GTPCH activity is subject to BH4-dependent feedback inhibition, via a mechanism that involves the formation of an inhibitory complex that comprises BH4, GTPCH and an auxiliary protein termed GFRP (for GTPCH feedback regulatory protein). Stimulation of GTPCH activity can be elicited by phenylalanine (Phe) and this also appears to require complex formation with GFRP. Despite potential importance, little is known regarding the allosteric mechanisms by which GFRP mediates inhibitory or stimulatory effects on GTPCH activity or the function of GFRP as an in vivo regulator of intracellular BH4 synthesis and NO production.; To gain insight into the molecular details of GTPCH allosteric regulation, an in vitro system to reconstitute feedback inhibition was established. This system uses a novel real-time kinetic assay as a readout for GTPCH activity. Distinct functional complexes of GTPCH/GFRP were discovered as defined by Km, Vmax and bound allosteric effector. Additionally, a mutant GFRP disabled in its ability to bind Phe was engineered and revealed that a failure to effectively bind Phe is associated with failure to engage potent feedback inhibition of GTPCH. It was also revealed that inorganic phosphate and pyrophosphate are substrate binding analogs for GTPCH, acting as allosteric activators or inhibitors and enhancing binding of GFRP.; The in vivo role of GFRP was studied by examining patterns of GTPCH protein expression in cells and tissues, and by analyzing BH4 and NO production in cells when GFRP protein levels were made deficient. Western blot analyses revealed that in some tissues and cells, GFRP is down-regulated by immunoactivators that enhance BH4 levels and NO synthesis. The extent to which GFRP influences GTPCH activity was directly determined using RNA interference to suppress GFRP gene expression in murine endothelial cells. Knock-down of GFRP protein resulted in a significant increase in basal BH4 levels, with a corresponding increase in NO production, indicating that GFRP tonically inhibits GTPCH activity in endothelial cells. Together, these findings indicate that GFRP functions as an important determinant of in vivo BH4 levels in NO-producing cells.