GTP Cyclohydrolase I (GCH1) catalyses the conversion of guanosine triphosphate (GTP) to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). BH4 functions as co-factor in neurotransmitter biosynthesis. BH4 homeostasis is a promising target to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). Dependent on the relative cellular concentrations of effector ligands, BH4 and phenylalanine, GFRP binds GCH1 to form inhibited or activated complexes, respectively. We determined high-resolution structures of the ligand-free and -bound human GFRP and GCH1-GFRP complexes by X-ray crystallography. Highly similar binding modes of the substrate analogue 7-deaza-GTP to active and inhibited GCH1-GFRP complexes confirm a novel, dissociation rate-controlled mechanism of non-competitive inhibition to be at work. Further, analysis of all structures shows that upon binding of the effector molecules, the conformations of GCH1 or GFRP are altered and form highly complementary surfaces triggering a picomolar interaction of GFRP and GCH1 with extremely slow k_off values, while GCH1-GFRP complexes rapidly disintegrate in absence of BH4 or phenylalanine. Finally, comparing behavior of full-length and N-terminally truncated GCH1 we conclude that the disordered GCH1 N-terminus does not have impact on complex formation and enzymatic activity. In summary, this comprehensive and methodologically diverse study helps to provide a better understanding of the regulation of GCH1 by GFRP and could thus stimulate research on GCH1 modulating drugs.

GTP 环化水解酶 I (GCH1) 催化三磷酸鸟苷 (GTP) 转化为三磷酸二氢蝶呤 (H2NTP)，这是四氢生物蝶呤 (BH4) 生物合成的起始步骤。BH4 在神经递质生物合成中起辅助因子的作用。BH4 稳态是治疗患者疼痛障碍的有希望的目标。哺乳动物 GCH1 的功能受代谢传感机制的调节，该机制涉及调节蛋白 GCH1 反馈调节蛋白 (GFRP)。根据效应配体 BH4 和苯丙氨酸的相对细胞浓度，GFRP 与 GCH1 结合，分别形成抑制或激活的复合物。我们通过 X 射线晶体学确定了无配体和结合的人 GFRP 和 GCH1-GFRP 复合物的高分辨率结构。底物类似物 7-deaza-GTP 与活性和受抑制的 GCH1-GFRP 复合物的高度相似的结合模式证实了一种新型的、解离速率控制的非竞争性抑制机制正在发挥作用。此外，对所有结构的分析表明，在效应分子结合后，GCH1 或 GFRP 的构象发生改变并形成高度互补的表面，触发 GFRP 和 GCH1 与极慢的 k 的皮摩尔相互作用_off值，而 GCH1-GFRP 复合物在没有 BH4 或苯丙氨酸的情况下会迅速分解。最后，比较全长和 N 端截短的 GCH1 的行为，我们得出结论，无序的 GCH1 N 端对复合物的形成和酶活性没有影响。总之，这项全面且方法多样的研究有助于更好地了解 GFRP 对 GCH1 的调节，从而刺激对 GCH1 调节药物的研究。