Role of human GTP cyclohydrolase I and its regulatory protein in tetrahydrobiopterin metabolism

Abstract.

Objective: GTP cyclohydrolase I (GTPCH I) catalyzes the de novo biosynthesis of tetrahydrobiopterin (BH₄), an essential cofactor of NO-synthase. The enzyme underlies negative feedback regulation by the end product BH₄. This feedback inhibition is mediated through complex formation with the GTP cyclohydrolase I feedback regulatory protein (GFRP). To further classify the mechanism involved in the regulation of BH₄ synthesis, we measured expression of GTPCH I and GFRP in different human tissues. Furthermore, we looked for the influence of phenylalanine that is known to reverse BH₄-mediated feedback inhibition of GTPCH I, and of immunostimulation with interferon γ on the expression of GTPCH I and GFRP. Methods and results: Using RT-PCR and northern blot technique, coexpression of GFRP and GTPCH I could be demonstrated in a number of different tissues such as endothelial cells and peripheral blood cells. Following stimulation of human umbilical vein endothelial cells (HUVEC) with phenylalanine (1 mM), there was no change of GFRP mRNA. In contrast, the mRNA level of GTPCH I was significantly upregulated with a maximum after 6 hours (p = 0.04). Incubation of HUVEC with interferon-γ (100 U/ml) showed an increase of GTPCH I mRNA and a significant downregulation of GFRP mRNA after 24 hours (p = 0.03). Conclusion: This study shows for the first time the expression of GFRP in different human tissues. The biosynthesis of BH₄ is not only regulated on the substrate level but also through transcription of the interacting proteins. Phenylalanine stimulates the biosynthesis of BH₄ not only by reversing the negative feedback inhibition of GTPCH I but also by increasing the mRNA level of GTPCH I. Immunostimulation alters protein expression of GTPCH I and GFRP in a way that favors BH₄ synthesis.