GTP Cyclohydrolase I Feedback Regulatory Protein Is a Pentamer of Identical Subunits

Abstract

GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by tetrahydrobiopterin and also mediates the stimulatory effect of phenylalanine on the enzyme activity. To characterize the molecular structure of GFRP, we have purified it from rat liver using an efficient step of affinity chromatography and isolated cDNA clones, based on partial amino acid sequences of peptides derived from purified GFRP. Comparison between the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of purified GFRP showed that the mature form of GFRP consists of 83 amino acid residues with a calculated M r of 9,542. The isolated GFRP cDNA was expressed in Escherichia coli as a fusion protein with six consecutive histidine residues at its N terminus. The fusion protein was affinitypurified and digested with thrombin to remove the histidine tag. The resulting recombinant GFRP showed kinetic properties similar to those of GFRP purified from rat liver. Cross-linking experiments using dimethyl suberimidate indicated that GFRP was a pentamer of 52 kDa. Sedimentation equilibrium measurements confirmed the pentameric structure of GFRP by giving an average M r of 49,734, which is 5 times the calculated molecular weight of the recombinant GFRP polypeptide. Based on the pentameric structure of GFRP, we have proposed a model for the quaternary structure of GFRP and GTP cyclohydrolase I complexes. 6,7, 1 is an essential cofactor for aromatic amino-acid hydroxylases and nitric-oxide synthases (1, 2). The intracellular level of BH 4 is subsaturating for these enzyme reactions and thus is thought to be an important regulator of the activities of these enzymes. BH 4 is synthesized from GTP by a pathway composed of four enzyme reactions (1). Most important for regulation of BH 4 biosynthesis is GTP cyclohydrolase I (EC 3.5.4.16), which catalyzes the first and rate-limiting step of the conversion of GTP to dihydroneopterin triphosphate. GTP cyclohydrolase I is present in many organisms, ranging from bacteria to animal. In bacteria, this enzyme functions as the first enzyme in the biosynthesis of folic acid and not of BH 4 (1). Rat GTP cyclohydrolase I is composed of multiple identical subunits and shows positive cooperativity against GTP (3, 4).We have recently reported the identification of a new regulatory protein (GFRP) which mediates the feedback inhibition of GTP cyclohydrolase I by the end product of this pathway, BH 4 (5). GFRP and BH 4 inhibit the enzyme activity of GTP cyclohydrolase I by decreasing its maximum velocity while having little effect on the affinity of GTP, indicating noncompetitive inhibition. GFRP exerts its inhibitory effect on GTP cyclohydrolase I by forming a complex with GTP cyclohydrolase I in the presence of BH 4 and GTP. Formation of the complex between the two proteins is reversible, depending on the presence of BH 4 and GTP. Furthermore, the inhibition of GTP cyclohydrolase I by GFRP and BH 4 is specifically reversed by L-phenylalanine. ...

Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP

“…Rat recombinant GTPCHI and GFRP were expressed in Escherichia coli cells, purified, and crystallized as described (13,16,17). The stimulatory complex (5 mg͞ml) was crystallized from 24% (vol/vol) 2-methyl-2,4-pentanediol͞75 mM Tris⅐HCl (pH 7.5)͞50 mM KCl͞5 mM phenylalanine.…”

“…GTPCHI is a decamer of 260 kDa with a subunit consisting of 230 amino acid residues (15), and GFRP is a pentamer of 50 kDa with a subunit consisting of 83 amino acid residues (16). Recently, we showed that both stimulatory and inhibitory GTPCHI⅐GFRP complexes consist of one molecule of decameric GTPCHI and two molecules of pentameric GFRP (13).…”

The Mechanism of Potent GTP Cyclohydrolase I Inhibition by 2,4-Diamino-6-hydroxypyrimidine

“…Interestingly, millimolar level concentrations of L-Phe have been shown to reverse this feedback inhibition without causing dissociation of the GFRP⅐GTPCH complex (37). In the absence of BH4, L-Phe triggers GFRP binding to GTPCH resulting in increased GTPCH activity at low levels of GTP substrate (35,38). Because L-Phe is potentially neurotoxic, the ability of L-Phe to stimulate GTPCH activity may have evolved to provide an abundant supply of BH4 cofactor for optimal activity of phenylalanine hydroxylase.…”

GTP cyclohydrolase I utilizes metal‐free GTP as its substrate

“…At the post-transcriptional level, BH 4 was shown to inhibit, and phenylalanine to stimulate, GCH activity through interaction with GFRP, a GTP cyclohydrolase I feedback regulatory protein [9]. GCH, which is a homodecameric protein, shows positive cooperativity against the GTP substrate [10] and phenylalanine changes the substrate velocity curve from sigmoidal to hyperbolic [11].Recent biophysical studies suggest a stimulatory effect of Zn 2+ [12] and Ca 2+ [13] on GCH activity. By crystallographic analysis using purified Escherichia coli enzyme [14], an N-terminally truncated form of the recombinant human enzyme [12], and a stimulatory complex of rat GCH and GFRP induced by phenylalanine [15], Zn 2+ was shown to be bound to the active centre of the homodecameric GCH enzyme.…”

“…At the post-transcriptional level, BH 4 was shown to inhibit, and phenylalanine to stimulate, GCH activity through interaction with GFRP, a GTP cyclohydrolase I feedback regulatory protein [9]. GCH, which is a homodecameric protein, shows positive cooperativity against the GTP substrate [10] and phenylalanine changes the substrate velocity curve from sigmoidal to hyperbolic [11].…”