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

Inhibition of GTP cyclohydrolase I (GTPCH) has been used as a selective tool to assess the role of de novo synthesis of (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4) in a biological system. Toward this end, 2,4-diamino-6-hydroxypyrimidine (DAHP) has been used as the prototypical GTPCH inhibitor. Using a novel real-time kinetic microplate assay for GTPCH activity and purified prokaryote-expressed recombinant proteins, we show that potent inhibition by DAHP is not the result of a direct interaction with GTPCH. Rather, inhibition by DAHP in phosphate buffer occurs via an indirect mechanism that requires the presence of GTPCH feedback regulatory protein (GFRP). Notably, GFRP was previously discovered as the essential factor that reconstitutes inhibition of pure recombinant GTPCH by the pathway end product BH4. Thus, DAHP inhibits GTPCH by engaging the endogenous feedback inhibitory system. We further demonstrate that l-Phe fully reverses the inhibition of GTPCH by DAHP/GFRP, which is also a feature in common with inhibition by BH4/GFRP. These findings suggest that DAHP is not an indiscriminate inhibitor of GTPCH in biological systems; instead, it is predicted to preferentially attenuate GTPCH activity in cells that most abundantly express GFRP and/or contain the lowest levels of l-Phe. Inhibition of GTP cyclohydrolase I (GTPCH) has been used as a selective tool to assess the role of de novo synthesis of (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4) in a biological system. Toward this end, 2,4-diamino-6-hydroxypyrimidine (DAHP) has been used as the prototypical GTPCH inhibitor. Using a novel real-time kinetic microplate assay for GTPCH activity and purified prokaryote-expressed recombinant proteins, we show that potent inhibition by DAHP is not the result of a direct interaction with GTPCH. Rather, inhibition by DAHP in phosphate buffer occurs via an indirect mechanism that requires the presence of GTPCH feedback regulatory protein (GFRP). Notably, GFRP was previously discovered as the essential factor that reconstitutes inhibition of pure recombinant GTPCH by the pathway end product BH4. Thus, DAHP inhibits GTPCH by engaging the endogenous feedback inhibitory system. We further demonstrate that l-Phe fully reverses the inhibition of GTPCH by DAHP/GFRP, which is also a feature in common with inhibition by BH4/GFRP. These findings suggest that DAHP is not an indiscriminate inhibitor of GTPCH in biological systems; instead, it is predicted to preferentially attenuate GTPCH activity in cells that most abundantly express GFRP and/or contain the lowest levels of l-Phe. (6R)-5,6,7,8-Tetrahydro-l-biopterin (BH4) 1The abbreviations used are: BH4, (6R)-5,6,7,8-tetrahydro-l-biopterin; GTPCH, GTP cyclohydrolase; DAHP, 2,4-diamino-6-hydroxypyrimidine; GFRP, GTPCH feedback regulatory protein; IPTG, isopropyl-l-thio-β-d-galactopyranoside; MBP, maltose-binding protein; H2NTP, dihydroneopterin triphosphate; HPLC, high pressure liquid chromatography. is an essential cofactor for phenylalanine hydroxylase (1Kaufman S. Proc. Natl. Acad. Sci. U. S. A. U. S. A. 1963; 50: 1085-1093Crossref PubMed Scopus (347) Google Scholar), tyrosine hydroxylase (2Nagatsu T. Levitt M. Udenfriend S. J. Biol. Chem. 1964; 139: 2910-2917Abstract Full Text PDF Google Scholar), tryptophan hydroxylase (3Lovenberg W. Jequier E. Sjoerdsma A. Science. 1967; 155: 217-219Crossref PubMed Scopus (375) Google Scholar), glycerol ether monoxygenases (4Kaufman S. Pollock R.J. Summer G.K. Das A.K. Hajra A.K. Biochim. Biophys. Acta. 1990; 1040: 19-27Crossref PubMed Scopus (41) Google Scholar, 5Tiets A. Lindberg M. Kennedy E. J. Biol. Chem. 1964; 239: 4081-4090Abstract Full Text PDF PubMed Google Scholar), and the three isoforms of nitric-oxide synthase (6Tayeh M.A. Marletta M.A. J. Biol. Chem. 1989; 264: 19654-19658Abstract Full Text PDF PubMed Google Scholar, 7Kwon N.S. Nathan C.F. Stuehr D.J. J. Biol. Chem. 1989; 264: 20496-20501Abstract Full Text PDF PubMed Google Scholar). Because the activity of these enzymes is typically limited by the availability of BH4, intracellular levels of BH4 determine the rate of production of several key cell-signaling molecules including nitric oxide (NO), dopamine, norepinephrine, epinephrine, and serotonin. In mammalian cells, BH4 levels are primarily dictated by the activity of GTP cyclohydrolase I (GTPCH), the first of three enzymes in the de novo BH4 synthesis pathway (8Nichol C.A. Smith G.K. Duch D.S. Annu. Rev. Biochem. 1985; 54: 729-764Crossref PubMed Google Scholar). Mutations in the GTPCH gene are responsible for severe diseases including 3,4-dehydroxyphenylalanine (dopa)-responsive dystonia (9Ichinose H. Ohye T. Takahashi E. Seki N. Hori T. Segawa M. Nomura Y. Endo K. Tanaka H. Tsuji S. Nat. Genet. 1994; 8: 236-242Crossref PubMed Scopus (715) Google Scholar) and atypical phenylketonuria (10Thony B. Blau N. Hum. Mutat. 1997; 10: 11-20Crossref PubMed Scopus (68) Google Scholar). Other neurological disorders, including Parkinson's disease (11Curtius H.C. Niederwieser A. Levine R. Muldner H. Adv. Neurol. 1984; 40: 463-466PubMed Google Scholar), Alzheimer's disease (12Barford P.A. Blair J.A. Eggar C. Hamon C. Morar C. Whitburn S.B. J. Neurol. Neurosurg. Psychiatry. 1984; 47: 736-738Crossref PubMed Scopus (69) Google Scholar, 13Lovenberg W. Levine R.A. Robinson D.S. Ebert M. Williams A.C. Calne D.B. 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Cardiol. 2000; 35: 71-75Crossref PubMed Scopus (135) Google Scholar), and hypercholesterolemia (20Stroes E. Kastelein J. Cosentino F. Erkelens W. Wever R. Koomans H. Luscher T. Rabelink T. J. Clin. Investig. 1997; 99: 41-46Crossref PubMed Scopus (514) Google Scholar), are all associated with deficient BH4 levels. The prototypical GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP) has been used extensively to assess the roles of BH4 and GTPCH in physiological and pathophysiological conditions, although the exact mechanism of this inhibition has not been defined. Four decades ago, DAHP was identified as a potent inhibitor of biopterin-dependent growth of the protozoan Crithidia fasciculata (21Rembold H. Hoppe Seyler's Z Physiol. Chem. 1964; 339: 258-259Crossref PubMed Scopus (5) Google Scholar). Investigations of biopterin excretion in rats suggested the presence of a de novo biopterin synthesis pathway that is potently inhibited following DAHP treatment (22Pabst W. Rembold H. Hoppe Seyler's Z Physiol. Chem. 1966; 344: 107-112Crossref PubMed Scopus (15) Google Scholar). Subsequently, biosynthesis of 7,8-dihydrobiopterin from GTP was demonstrated in rat brain, and the responsible multienzyme pathway was found to be inhibited by DAHP (23Gal E.M. Nelson J.M. Sherman A.D. Neurochem. Res. 1978; 3: 69-88Crossref PubMed Scopus (79) Google Scholar, 24Gal E.M. Sherman A.D. Neurochem. Res. 1976; 1: 627-639Crossref PubMed Scopus (25) Google Scholar). DAHP-induced BH4 deficiency was later used to generate an animal model for hyperphenylalaninemia (25Cotton R.G. J. Inherit. Metab. Dis. 1986; 9: 4-14Crossref PubMed Scopus (15) Google Scholar) as well as a model for neurological BH4 deficiency (26Suzuki S. Watanabe Y. Tsubokura S. Kagamiyama H. Hayaishi O. Brain Res. 1988; 446: 1-10Crossref PubMed Scopus (41) Google Scholar). Additionally, DAHP has been widely used to determine the impact of BH4 availability on NO synthesis in cells in culture (27Gross S.S. Jaffe E.A. Levi R. Kilbourn R.G. Biochem. Biophys. Res. Commun. 1991; 178: 823-829Crossref PubMed Scopus (371) Google Scholar, 28Jorens P.G. van Overveld F.J. Bult H. Vermeire P.A. Herman A.G. Br. J. Pharmacol. 1992; 107: 1088-1091Crossref PubMed Scopus (8) Google Scholar, 29Gross S.S. Levi R. J. Biol. Chem. 1992; 267: 25722-25729Abstract Full Text PDF PubMed Google Scholar, 30Oddis C.V. Finkel M.S. Am. J. Physiol. 1996; 270: H1864-H1868Crossref PubMed Google Scholar, 31Saura M. Perez-Sala D. Canada F.J. Lamas S. J. Biol. Chem. 1996; 271: 14290-14295Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 32Delgado-Esteban M. Almeida A. Medina J.M. J. Neurochem. 2002; 82: 1148-1159Crossref PubMed Scopus (50) Google Scholar) and in animals (33Mitchell B.M. Dorrance A.M. Webb R.C. Am. J. Physiol. 2003; 285: H2165-H2170Google Scholar, 34Bune A.J. Brand M.P. Heales S.J. Shergill J.K. Cammack R. Cook H.T. Biochem. Biophys. Res. Commun. 1996; 220: 13-19Crossref PubMed Scopus (48) Google Scholar). Because of the overt structural homology between DAHP and the GTPCH substrate GTP, the ability of DAHP to inhibit GTPCH has been assumed to result from competition for substrate binding. However, DAHP is also structurally similar to BH4 (Fig. 1) and, inasmuch as GTPCH is regulated by end product feedback inhibition, DAHP could potentially inhibit GTPCH activity by co-opting the endogenous feedback inhibitory mechanism. Feedback inhibition by BH4 is mediated by the formation of a ternary complex between BH4, GTPCH, and a protein termed GTPCH feedback regulatory protein (GFRP) (35Harada T. Kagamiyama H. Hatakeyama K. Science. 1993; 260: 1507-1510Crossref PubMed Scopus (136) Google Scholar, 36Milstien S. Jaffe H. Kowlessur D. Bonner T.I. J. Biol. Chem. 1996; 271: 19743-19751Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). GTPCH is a stable decamer, and in the presence of BH4, feedback inhibition is engaged by the binding of one GFRP pentamer to each of the two pentameric faces of GTPCH. Interestingly, millimolar level concentrations of l-Phe have been shown to reverse this feedback inhibition without causing dissociation of the GFRP·GTPCH complex (37Yoneyama T. Hatakeyama K. J. Biol. Chem. 1998; 273: 20102-20108Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In the absence of BH4, l-Phe triggers GFRP binding to GTPCH resulting in increased GTPCH activity at low levels of GTP substrate (35Harada T. Kagamiyama H. Hatakeyama K. Science. 1993; 260: 1507-1510Crossref PubMed Scopus (136) Google Scholar, 38Yoneyama T. Brewer J.M. Hatakeyama K. J. Biol. Chem. 1997; 272: 9690-9696Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). 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. A previous observation that DAHP-mediated inhibition of GTPCH in rat aortic smooth muscle cells is partially reversed by addition of l-Phe suggests that the mode of inhibition by DAHP may be GFRP-dependent (39Xie L. Smith J.A. Gross S.S. J. Biol. Chem. 1998; 273: 21091-21098Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Nonetheless, DAHP has been widely assumed to selectively inhibit GTPCH activity via competition for binding substrate GTP. Utilizing a novel kinetic assay for GTPCH activity and purified recombinant rat GTPCH and GFRP from prokaryotic expression systems, we have now conducted a rigorous in vitro analysis of the mechanism of GTPCH inhibition by DAHP. Results confirm that potent GTPCH inhibition by DAHP requires GFRP and recapitulates the endogenous mechanism by which BH4 inhibits its own synthesis. Expression and Purification of Recombinant GFRP—GFRP cDNA was amplified by PCR from a rat aortic smooth muscle cell cDNA library (Invitrogen). To facilitate insertion into the pET15b expression vector (Novagen), primers for PCR were designed to include the desired restriction enzyme cut sites. The GFRP forward primer (5′-CCAGCCACTCCATATGCCCTAAA-3′) contained a site for NdeI digestion, and the GFRP reverse primer (5′-AAGTCAGCTCACTCGAGTCATTCC-3′) contained an XhoI cut site. PCR was performed by standard methods using 2 μl of DNA template and the following schedule: 95 °C for 5 min followed by 35 cycles of denaturation (94 °C for 45 s), annealing (55 °C for 1 min), and elongation (72 °C for 1 min), terminating with extension at 72 °C for 10 min. The resulting PCR products were separated on a 1% agarose gel containing ethidium bromide and visualized by UV fluorescence. The predicted GFRP cDNA-containing product (255 bp) was observed and gel-purified (Bio 101, GeneClean). The purified PCR product and pET15b plasmid were both digested with NdeI and XhoI and resolved by electrophoresis. Following repurification from the gel, the GFRP insert was ligated into the plasmid using T4 DNA ligase (Invitrogen) at 24 °C overnight. On insertion of GFRP cDNA between the NdeI and XhoI sites, the resulting pET15b expression construct encodes a stretch of six His residues fused to the N terminus of GFRP. Competent DH5α Escherichia coli (Invitrogen) were transformed with the ligation product, and positive clones were selected using LB agar plates containing ampicillin of GFRP cDNA in pET15b was by restriction enzyme DNA from a positive was purified and used to the E. coli expression A transformed with the expression construct was in LB containing ampicillin and at °C with at the an of expression of the GFRP protein was with 1 at in the for were by and the resulting was at °C were and in buffer 5 5 containing 1 were by and the was by at The was to an of The was with of buffer without followed by of buffer was from the with buffer were for by and protein containing a at the predicted of the protein were and to buffer using The protein was into a buffer containing The presence of in this buffer was to of Expression and Purification of Recombinant rat GTPCH was as a maltose-binding protein protein and purified from E. coli as previously (39Xie L. Smith J.A. Gross S.S. J. Biol. Chem. 1998; 273: 21091-21098Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In DH5α E. coli were transformed with a expression plasmid to express expression was by in LB with 1 at °C for were by and by The was by and to a of for of was from the with 10 and for using and protein The purified protein a level of GTPCH activity at of for GTPCH of dihydroneopterin the product of GTPCH, was using a kinetic assay M.A. Gross S.S. Blau N. B. and in Scholar). A of the desired of the following phosphate buffer GTPCH, 10 GTP, DAHP, and The of was from the rate of in at a at °C using a microplate a of were used for of the GTPCH activity of and for is to were by of the desired concentrations of GTP and a of GTPCH M.A. Gross S.S. Blau N. B. and in Scholar). the of was to the of GTP, were to generate a standard was used to a of for the The and were used to a between and GTPCH The for GTP was also and used to determine the to which of GTP with the of by GTP was to be of GTP the the to by of the in for GTPCH activity of GTPCH activity was by at °C as for the microplate assay and a was for product analysis using the assay for Gross S.S. M. in Scholar). To this end, of in the was by treatment with and 10 for 1 at °C in the were with 10 μl of with μl of 1 and for 1 at °C by treatment with μl of in a at were to reverse high liquid and was by with a pure and LB were from BH4 was from DAHP, GTP, and all were from and were of the were by of a for GTPCH has a the substrate GTP A. N. A. 40: PubMed Scopus Google Scholar), and this feature was used for the of a high kinetic microplate assay for GTPCH activity M.A. Gross S.S. Blau N. B. and in Scholar). in was a of and GTP in the presence of a of recombinant GTPCH enzyme (Fig. was also on GTPCH and, a was for at min substrate GTP rate (Fig. A standard was on of GTP to (Fig. as Notably, a was between and production (Fig. of the standard were by the of substrate concentrations to product by a of GTPCH the standard from the in A is The a between and an of GTPCH of GTPCH in the of DAHP that Inhibition the to which DAHP inhibits GTPCH activity via competition for substrate recombinant rat GTPCH activity was a of GTP concentrations in the presence and absence of DAHP, a that inhibits BH4 synthesis in cells (Fig. (39Xie L. Smith J.A. Gross S.S. J. Biol. Chem. 1998; 273: 21091-21098Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In the absence of DAHP, the and for GTP were and in with (39Xie L. Smith J.A. Gross S.S. J. Biol. Chem. 1998; 273: 21091-21098Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, A. A. M. M. M. M. J. H. R. A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar, K. T. S. Watanabe Y. Kagamiyama H. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar). On addition of DAHP, the for GTP was increased and was as by The DAHP-induced in was to the potent of BH4 synthesis observed with DAHP in Inhibition of GTPCH by DAHP an GFRP-dependent on the structural between DAHP and BH4 it was that DAHP may be as a to feedback inhibition of GTPCH (39Xie L. Smith J.A. Gross S.S. J. Biol. Chem. 1998; 273: 21091-21098Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). To this recombinant GFRP was purified from a prokaryotic expression (Fig. and with of the feedback inhibition in GTPCH activity was in the presence of a GTP with concentrations of DAHP in the presence absence of GFRP. shown in DAHP a and inhibition of GTPCH in the presence of GFRP, in with a mechanism of GTPCH Notably, GTPCH activity was by with DAHP the addition of GFRP increased inhibition to of GTPCH inhibition by GFRP in vitro suggests that DAHP the feedback inhibitory for potent inhibition of GTPCH in biological of GFRP on the ability of DAHP to inhibit GTPCH. The activity of recombinant rat GTPCH was using the assay as The inhibitory of DAHP was in the absence presence of recombinant rat GFRP The of l-Phe in with GFRP was also that inhibition of GTPCH by DAHP is in the presence of GFRP. of inhibition by l-Phe is in with a GFRP-dependent mechanism to that for feedback inhibition by BH4. are from two each using for each of Inhibition by potent inhibition of GTPCH was DAHP and GFRP. The addition of concentrations of l-Phe to GTPCH in of activity (Fig. The of l-Phe for of GTPCH inhibition was Notably, of GTPCH inhibition was found with at concentrations to A. and S. S. GFRP-dependent inhibition of GTPCH has been found to be reversed by l-Phe and by (35Harada T. Kagamiyama H. Hatakeyama K. Science. 1993; 260: 1507-1510Crossref PubMed Scopus (136) Google Scholar, 36Milstien S. Jaffe H. Kowlessur D. Bonner T.I. J. Biol. Chem. 1996; 271: 19743-19751Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). of the GFRP and l-Phe of GTPCH Inhibition by is a that to as the microplate assay for GTPCH activity on of for we the of an of a DAHP-induced in Thus, we to that GFRP is for inhibition of GTPCH by DAHP using an assay Results shown in confirm that DAHP inhibition is GFRP-dependent and as demonstrated by the assay for GTPCH on and of in The in by the assay with the microplate assay (Fig. is observed and to in for analysis and The of GFRP to GTPCH the of DAHP as a GTPCH the GFRP of GTPCH inhibition by DAHP, the activity of a of GTPCH was in the presence of DAHP and of GFRP. shown in the of DAHP was as the of GFRP to GTPCH increased a of at which inhibition was These the that the of GFRP protein expression the of GTPCH inhibition by DAHP in cells and We a novel kinetic assay for and of GTPCH activity in assay and formation of in on GTPCH The microplate is with to and for high analysis of GTPCH activity A is that the assay be used with pure partially purified GTPCH of by in biological not of the assay to the mechanism for potent inhibition of GTPCH by DAHP. DAHP is a inhibitor of GTPCH activity and has been widely used to inhibit de novo BH4 synthesis in biological of DAHP as an inhibitor of BH4 the mechanism of inhibition has been it has been assumed that inhibition is on a structural of DAHP with the GTPCH GTP. is that in GTPCH activity is to feedback inhibition by the pathway end product BH4. The mechanism of feedback inhibition the formation of an inhibitory complex between GTPCH and the regulatory protein GFRP. l-Phe reverses this inhibition without the inhibitory The structural of DAHP and BH4 as well as from previous conducted in rat aortic smooth muscle cells (39Xie L. Smith J.A. Gross S.S. J. Biol. Chem. 1998; 273: 21091-21098Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) and in vitro T. Hatakeyama K. Biochem. Biophys. PubMed Scopus Google Scholar) suggested a role for GFRP in the mechanism of DAHP-mediated on using pure recombinant proteins, findings confirm that a mode of DAHP inhibition of GTPCH is on GFRP. In the presence of GFRP, inhibition of GTPCH by DAHP was observed to be and reversed by which are that the GFRP-dependent GTPCH feedback inhibitory system. GTPCH were performed in phosphate buffer Interestingly, the mode of inhibition of GTPCH by DAHP was in a buffer it was found to be Thus, in to the inhibition of GTPCH could be observed in the absence of GFRP and was not found to be reversed by l-Phe However, a B.M. Dorrance A.M. Webb R.C. J. Pharmacol. 43: PubMed Scopus Google Scholar) that treatment of rats with DAHP in a in pressure by a mechanism that was reversed on with in with the that in inhibition of GTPCH by DAHP is The observation of GFRP-dependent inhibition of GTPCH by DAHP in phosphate buffer may be by the binding of phosphate to the GTPCH the binding of DAHP as a inhibitor and the mode of inhibition to of E. coli GTPCH phosphate that with the of the GTP J. A. H. C. N. J. A. R. M. J. Biol. 2003; PubMed Scopus (65) Google Scholar). The of physiological levels of phosphate to GTPCH and interaction with GFRP the model in biological conditions, DAHP is as a to feedback inhibition of GTPCH. These may in in the of DAHP as an inhibitor of BH4 synthesis in The of DAHP on the cell of GFRP and l-Phe. We for with the assay for GTPCH

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