Abstract
Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets 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). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1-GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.
Highlights
Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4)
Abnormalities in the control mechanisms of GCH1 or the activities in other enzymes of its biosynthetic pathway leads to BH4 deficiency, which is linked to a variety of vascular diseases such as diabetes, atherosclerosis, and hypertension [9,10,11,12,13,14] and to neurological disorders, including Parkinson’s disease [15, 16]
The effects of GCH1 feedback regulatory protein (GFRP) on GCH1 occur via formation of heteromeric protein complexes between GCH1 and GFRP, which are dependent on the intracellular concentrations of the effector molecules phenylalanine or BH4
Summary
Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Abnormalities in the control mechanisms of GCH1 or the activities in other enzymes of its biosynthetic pathway leads to BH4 deficiency, which is linked to a variety of vascular diseases such as diabetes, atherosclerosis, and hypertension [9,10,11,12,13,14] and to neurological disorders, including Parkinson’s disease [15, 16]. These examples impressively show the serious consequences of nonphysiological levels of BH4. We propose a dissociation rate-controlled mechanism of allosteric, noncompetitive inhibition, which could stimulate further research toward the development of GCH1 inhibitors to treat neuropathic and inflammatory pain disorders
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