Tetrahydrobiopterin biosynthetic pathway and deficiency

It has been proven that the most common defect in the tetrahydrobiopterin biosynthesis is caused by 6-pyruvoyl tetrahydropterin synthase deficiency. The enzyme 6-pyruvoyl tetrahydropterin synthase consists of four identical subunits which convert dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin in the presence of magnesium. UV, NMR, and MS data prove that the enzyme catalyzes the elimination of triphosphate as well as the intramolecular rearrangement. The 6-pyruvoyl tetrahydropterin synthase activity was measured in fetal erythrocytes and together with the neopterin and biopterin measurements in amniotic fluid this enabled performing prenatal diagnosis of 6-pyruvoyl tetrahydropterin synthase deficiency. Peripheral tetrahydrobiopterin deficiency was shown to be due to an incomplete 6-pyruvoyl tetrahydropterin synthase deficiency or heterozygosity.     

Crystallographic and kinetic investigations on the mechanism of 6-pyruvoyl tetrahydropterin synthase

The enzyme 6-pyruvoyl tetrahydropterin synthase (PTPS) catalyses the second step in the de novo biosynthesis of tetrahydrobiopterin, the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin. The Zn and Mg-dependent reaction includes a triphosphate elimination, a stereospecific reduction of the N5-C6 double bond and the oxidation of both side-chain hydroxyl groups. The crystal structure of the inactive mutant Cys42Ala of PTPS in complex with its natural substrate dihydroneopterinetriphosphate was determined at 1.9 A resolution. Additionally, the uncomplexed enzyme was refined to 2.0 A resolution. The active site of PTPS consists of the pterin-anchoring Glu A107 neighboured by two catalytic motifs: a Zn(II) binding site and an intersubunit catalytic triad formed by Cys A42, Asp B88 and His B89. In the free enzyme the Zn(II) is in tetravalent co-ordination with three histidine ligands and a water molecule. In the complex the water is replaced by the two substrate side-chain hydroxyl groups yielding a penta-co-ordinated Zn(II) ion. The Zn(II) ion plays a crucial role in catalysis. It activates the protons of the substrate, stabilizes the intermediates and disfavours the breaking of the C1'C2' bond in the pyruvoyl side-chain. Cys A42 is activated by His B89 and Asp B88 for proton abstraction from the two different substrate side-chain atoms C1', and C2'. Replacing Ala A42 in the mutant structure by the wild-type Cys by modelling shows that the C1' and C2' substrate side-chain protons are at equal distances to Cys A42 Sgamma. The basicity of Cys A42 may be increased by a catalytic triad His B89 and Asp B88. The active site of PTPS seems to be optimised to carry out proton abstractions from two different side-chain C1' and C2' atoms, with no obvious preference for one of them. Kinetic studies with dihydroneopterin monophosphate reveal that the triphosphate moiety of the substrate is necessary for enzyme specifity.     

Three-dimensional structure of 6-pyruvoyl tetrahydropterin synthase, an enzyme involved in tetrahydrobiopterin biosynthesis.

The crystal structure of rat liver 6-pyruvoyl tetrahydropterin synthase has been solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 20.4% at 2.3 A resolution. 6-Pyruvoyl tetrahydrobiopterin synthase catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. The functional enzyme is a hexamer of identical subunits. The 6-pyruvoyl tetrahydropterin synthase monomer folds into a sequential, four-stranded, antiparallel beta-sheet with a 25 residue, helix-containing insertion between strands 1 and 2 at the bottom of the molecule, and a segment between strands 2 and 3 forming a pair of antiparallel helices, layered on one side of the beta-sheet. Three 6-pyruvoyl tetrahydropterin synthase monomers form an unusual 12-stranded antiparallel beta-barrel by tight association between the N- and C-terminal beta-strands of two adjacent subunits. The barrel encloses a highly basic pore of 6-12 A diameter. Two trimers associate in a head-to-head fashion to form the active enzyme complex. The substrate-binding site is located close to the trimer-trimer interface and comprises residues from three monomers: A, A' and B. A metal-binding site in the substrate-binding pocket is formed by the three histidine residues 23, 48 and 50 from one 6-pyruvoyl tetrahydropterin synthase subunit. Close to the metal, but apparently not liganding it, are residues Cys42, Glu133 (both from A) and His89 (from B), which might serve as proton donors and acceptors during catalysis.     

Purification and characterization of 6-pyruvoyl tetrahydropterin synthase from salmon liver

Salmon liver was chosen for the isolation of 6-pyruvoyl tetrahydropterin synthase, one of the enzymes involved in tetrahydrobiopterin biosynthesis. A 9500-fold purification was obtained and the purified enzyme showed two single bands of 16 and 17 kDa on SDS/PAGE. The native enzyme (68 kDa) consists of four subunits and needs free thiol groups for enzymatic activity as was shown by reacting the enzyme with the fluorescent thiol reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide. The enzyme is heat-stable up to 80 degrees C, has an isoelectric point of 6.0-6.3, and a pH optimum at 7.5. The enzyme is Mg2+ -dependent and has a Michaelis constant for its substrate dihydroneopterin triphosphate of 2.2 microM. The turnover number of the purified salmon liver enzyme is about 50 times as high as that of the enzyme purified from human liver. It does not bind to the lectin concanavalin A, indicating that it is free of mannose and glucose residues. Polyclonal antibodies raised against the purified enzyme in Balb/c mice were able to immunoprecipitate enzyme activity. The same polyclonal serum was not able to immunoprecipitate enzyme activity of human liver 6-pyruvoyl tetrahydropterin synthase, nor was any cross-reaction in ELISA tests seen.     

Synthesis, utilization, and structure of the tetrahydropterin intermediates in the bovine adrenal medullary de novo biosynthesis of tetrahydrobiopterin

The biosynthesis of two tetrahydropterin intermediates (H4pterin-1 and H4pterin-2), their conversion to tetrahydrobiopterin, and their overall chemical structures are described. A new high performance liquid chromatographic separation of these and other tetrahydropterins is also described. The biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate proceeds in the presence of the bovine adrenal medullary biosynthetic enzymes, Mg2+, NADPH. The biosynthesis of H4pterin-2 occurs under identical conditions, and the compound accumulates in the presence of 1 to 10 microM of N-acetylserotonin, an inhibitor of sepiapterin reductase. At higher concentrations of the inhibitor, the synthesis of H4pterin-2 is also inhibited, and H4pterin-1 accumulates. H4pterin-1 also accumulates in the absence of NADPH. In the presence of NADPH the biosynthetic enzymes convert both intermediates to tetrahydrobiopterin at rates which are greater than the rate of conversion of dihydroneopterin triphosphate to tetrahydrobiopterin. Electrochemical, UV/VIS, oxidation, and ionization properties identify the compounds as tetrahydropterins. The side chain structures of the compounds were determined by a combination of chemical means. The structures of the compounds are 6R-(1',2'-dioxopropyl)-tetrahydropterin (H4pterin-1) and 6R-(L-1'-hydroxy-2'-oxopropyl)-tetrahydropterin (H4pterin-2). The data indicate that the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate proceeds in three steps: 1) formation of H4pterin-1 in the presence of Mg2+, 2) NADPH-dependent conversion of H4pterin-1 to H4pterin-2, and 3) NADPH-dependent conversion of H4pterin-2 to tetrahydrobiopterin.     

Computer studies on the stereostructure and quantum chemical properties of 6-pyruvoyl tetrahydropterin, the key intermediate of tetrahydrobiopterin biosynthesis

The optimized geometry of the conformation of atoms constituting the 6-pyruvoyl tetrahydropterin molecule, the labile key intermediate of tetrahydrobiopterin biosynthesis, was obtained by molecular orbital calculations within the MINDO/3 framework. The stereostructure of the molecule showing the preferred mode for binding to sepiapterin reductase or pyruvoyl tetrahydropterin reductase was drawn in perspective. The resulting structure with the equatorial staggered configuration of the 6-1',2'-dioxopropyl (pyruvoyl) side chain indicated that O(1') and H(6) were located in the trans position around the C(6)-C(1') bond and that the two vicinal carbonyls in the side chain were fixed in the incomplete trans form. The calculation of atomic charges and LUMO coefficients of these carbonyls suggests that the C2'-carbonyl may be more reactive toward NADPH than the C1'-carbonyl in the enzymatic reaction.     

The biosynthesis of tetrahydrobiopterin in rat brain. Purification and characterization of 6-pyruvoyl tetrahydropterin (2'-oxo)reductase

An enzyme with 6-pyruvoyl tetrahydropterin (6PPH4) (2'-oxo)reductase activity was purified to near homogeneity from whole rat brains by a rapid method involving affinity chromatography on Cibacron blue F3Ga-agarose followed by high performance ion exchange chromatography and high performance gel filtration. The enzyme has a single subunit of Mr 37,000 and has a similar amino acid composition to previously described aldoketo reductases. The reductase activity is absolutely dependent on NADPH, will only catalyze the reduction of the C-2'-oxo group of 6PPH4, and is inactive towards the C-1'-oxo group. However, the enzyme also shows high activity towards nonspecific substrates, such as 4-nitrobenzaldehyde, phenanthrenequinone, and menadione. The role of this 6PPH4 reductase in the formation of tetrahydrobiopterin (BH4) was investigated. Measurements were made of the rate of conversion of 6PPH4, generated from dihydroneopterin triphosphate with purified 6PPH4 synthase, to BH4 in the presence of mixtures of pure sepiapterin reductase and the 6PPH4 (2'-oxo)reductase purified from rat brains. The results suggest that when sepiapterin reductase activity is limiting, a large proportion of BH4 synthesis proceeds through the 6-lactoyl intermediate. However, when sepiapterin reductase is not limiting, most of the BH4 is probably formed via reduction of the other mono-reduced intermediate which is produced from 6PPH4 by sepiapterin reductase alone.     

Immunological studies on the participation of 6-pyruvoyl tetrahydropterin (2'-oxo) reductase, an aldose reductase, in tetrahydrobiopterin biosynthesis

The NADPH-dependent reduction of the two carbonyl groups in the side chain of the first tetrahydropterin intermediate on the tetrahydrobiopterin biosynthetic pathway, 6-pyruvoyl tetrahydropterin, proceeds in a sequential manner whose order has not yet been resolved. Sepiapterin reductase can catalyze the reduction of both carbonyl groups starting with the 1'-oxo. 6-Pyruvoyl tetrahydropterin (2'-oxo) reductase, which has now been shown to be a member of the aldose reductase family, catalyzes the formation of only the 2'-hydroxy-1'-oxo intermediate which still requires sepiapterin reductase for final conversion to tetrahydrobiopterin. Inhibiting antibodies to the 2'-oxo reductase have been prepared and utilized to explore the distribution of this reductase in rat brain. The antiserum also maximally inhibited in vitro tetrahydrobiopterin synthesis in crude rat brain extracts by 60%, indicating that the majority of tetrahydrobiopterin biosynthesis in vivo may proceed via the 2'-hydroxy-1'-oxo intermediate. However, analogous experiments with rat liver extracts demonstrate that inhibition of the 2'-oxo reductase activity does not inhibit the conversion of 6-pyruvoyl tetrahydropterin to tetrahydrobiopterin, suggesting that tetrahydrobiopterin biosynthesis may proceed via different pathways in rat brain and liver.     

Purification and characterization of 6-pyruvoyl tetrahydropterin synthase from human pituitary gland.

6-Pyruvoyl tetrahydropterin synthase, the enzyme that catalyses the conversion of 7,8-dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, was purified 3,330-fold from human pituitary gland with an overall recovery of 30%. The native enzyme has a molecular mass of 68 kD and consists of four identical subunits of 16.5 kD. The pH optimum of the enzyme in Tris/HCl buffer is 7.5. The enzyme is dependent on Mg2+ and NADPH and has a Michaelis-Menten constant of 10 microM for its natural substrate, 7,8-dihydroneopterin triphosphate. The isoelectric point of the human enzyme is 4.3-4.6. The human pituitary gland enzyme is heat instable in contrast to the enzymes from human, rat and salmon liver, and Drosophila head. The amino acid composition showed remarkably high content of acidic amino acids Asp and Glu. The N-terminus was found to be blocked.     

Reaction mechanism of GTP cyclohydrolase I: single turnover experiments using a kinetically competent reaction intermediate

GTP cyclohydrolase I catalyses the transformation of GTP into dihydroneopterin 3'-triphosphate, which is the first committed precursor of tetrahydrofolate and tetrahydrobiopterin. The kinetically competent reaction intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone, was used as substrate for single turnover experiments monitored by multiwavelength photometry. The early reaction phase is characterized by the rapid appearance of an optical transient with an absorption maximum centred at 320. This species is likely to represent a Schiff base intermediate at the initial stage of the Amadori rearrangement of the carbohydrate side-chain. Deconvolution of the optical spectra suggested four linearly independent processes. A fifth reaction step was attributed to photodecomposition of the enzyme product. Pre-steady state experiments were also performed with the H179A mutant which can catalyse a reversible conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone but is unable to form the final product, dihydroneopterin triphosphate. Optical spectroscopy failed to detect any intermediate in the reversible reaction sequence catalysed by the mutant protein. The data obtained with the wild-type and mutant protein in conjunction with earlier quenched flow studies show that the enzyme-catalysed opening of the imidazole ring of GTP and the hydrolytic release of formate from the resulting formamide type intermediate are both rapid reactions by comparison with the subsequent rearrangement of the carbohydrate side-chain which precedes the formation of the dihydropyrazine ring of dihydroneopterin triphosphate.     

Ring opening is not rate-limiting in the GTP cyclohydrolase I reaction

GTP cyclohydrolase I catalyzes a mechanistically complex ring expansion affording dihydroneopterin triphosphate and formate from GTP. Single turnover quenched flow experiments were performed with the recombinant enzyme from Escherichia coli. The consumption of GTP and the formation of 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate, formate, and dihydroneopterin triphosphate were determined by high pressure liquid chromatography analysis. A kinetic model comprising three consecutive unimolecular steps was used for interpretations where the first intermediate, 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone 5'-triphosphate, was formed in a reversible reaction. The rate constant k(1) for the reversible opening of the imidazole ring of GTP was 0.9 s(-1), the rate constant k(3) for the release of formate from 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate was 2.0 s(-1), and the rate constant k(4) for the formation of dihydroneopterin triphosphate was 0.03 s(-1). Thus, the hydrolytic opening of the imidazole ring of GTP is rapid by comparison with the overall reaction.     

Tetrahydrobiopterin biosynthetic activities in human macrophages, fibroblasts, THP-1, and T 24 cells. GTP-cyclohydrolase I is stimulated by interferon-gamma, and 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are constitutively present

Interferon-gamma induces tetrahydrobiopterin biosynthesis in human cells and cell lines. Macrophages are peculiar in the formation of large amounts of neopterin derivatives as compared to tetrahydrobiopterin (Werner, E. R., Werner-Felmayer, G., Fuchs, D., Hausen, A., Reibnegger, G., and Wachter, H. (1989) Biochem J. 262, 861-866). Here we compare the impact of interferon-gamma treatment on activities of GTP-cyclohydrolase I (EC 3.5.4.16), 6-pyruvoyl tetrahydropterin synthase, and sepiapterin reductase (EC 1.1.1.153) in human peripheral blood-derived macrophages, normal dermal fibroblasts, THP-1 myelomonocytic cells, and the T 24 bladder transitional-cell carcinoma line. Upon interferon-gamma treatment, GTP-cyclohydrolase I activity is increased 7- to 40-fold, whereas 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase activities, which are constitutively present in all four investigated cells, remain unchanged. In fibroblasts and T 24 cells GTP cyclohydrolase I activity is the rate-limiting step of tetrahydrobiopterin biosynthesis. In macrophages and in THP-1 cells, however, the induced GTP cyclohydrolase I activity is higher than the 6-pyruvoyl tetrahydropterin synthase activity, leading to the accumulation of neopterin and neopterin phosphates.     

Crystal structure of 7,8-dihydroneopterin triphosphate epimerase.

BACKGROUND: Dihydroneopterin triphosphate (H2NTP) is the central substrate in the biosynthesis of folate and tetrahydrobiopterin. Folate serves as a cofactor in amino acid and purine biosynthesis and tetrahydrobiopterin is used as a cofactor in amino acid hydroxylation and nitric oxide synthesis. In bacteria, H2NTP enters the folate biosynthetic pathway after nonenzymatic dephosphorylation; in vertebrates, H2NTP is used to synthesize tetrahydrobiopterin. The dihydroneopterin triphosphate epimerase of Escherichia coli catalyzes the inversion of carbon 2' of H2NTP. RESULTS: The crystal structure of the homo-octameric protein has been solved by a combination of multiple isomorphous replacement, Patterson search techniques and cyclic averaging and has been refined to a crystallographic R factor of 18.8% at 2.9 A resolution. The enzyme is a torus-shaped, D4 symmetric homo-octamer with approximate dimensions of 65 x 65 A. Four epimerase monomers form an unusual 16-stranded antiparallel beta barrel by tight association between the N- and C-terminal beta strands of two adjacent subunits. Two tetramers associate in a head-to-head fashion to form the active enzyme complex. CONCLUSIONS: The folding topology, quaternary structure and amino acid sequence of epimerase is similar to that of the dihydroneopterin aldolase involved in the biosynthesis of the vitamin folic acid. The monomer fold of epimerase is also topologically similar to that of GTP cyclohydrolase I (GTP CH-1), 6-pyrovoyl tetrahydropterin synthase (PTPS) and uroate oxidase (UO). Despite a lack of significant sequence homology these proteins share a common subunit fold and oligomerize to form central beta barrel structures employing different cyclic symmetry elements, D4, D5, D3 and D2, respectively. Moreover, these enzymes have a topologically equivalent acceptor site for the 2-amino-4-oxo pyrimidine (2-oxo-4-oxo pyrimidine in uroate oxidase) moiety of their respective substrates.     

The enzymatic conversion of dihydroneopterin triphosphate to tripolyphosphate and 6-pyruvoyl-tetrahydropterin, an intermediate in the biosynthesis of other pterins in Drosophila melanogaster

The enzyme, previously called "sepiapterin synthase A," has been purified by approximately 700-fold from the heads of Drosophila melanogaster. This enzyme catalyzes the Mg2+-dependent conversion of 2-amino-4-oxo-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydrop teridine triphosphate (dihydroneopterin triphosphate or H2-NTP) to two products, one of which we have identified as tripolyphosphate. The other product is a phosphate-free, unstable compound which is an intermediate in the biosynthesis of several other naturally occurring pterins in Drosophila. This product is stable enough under anaerobic conditions to allow it to be characterized as 6-pyruvoyl-5,6,7,8-tetrahydropterin (6-pyruvoyl-H4-pterin). The 3-carbon side chain was identified as a pyruvoyl group on the basis of the susceptibility of the enzymatic product to reduction with tritiated sodium borohydride and the determination of the amounts and the sites of incorporation of tritium resulting from this reduction. From these observations, we suggest that this enzyme be renamed "6-pyruvoyl-H4-pterin synthase."     

Occurrence of GTP cyclohydrolase I in Bacillus stearothermophilus

A GTP cyclohydrolase which catalyzes the removal of carbon 8 of GTP as formic acid to yield a single pteridine compound occurs in an obligate thermophile Bacillus stearothermophilus ATCC 8005. The enzyme was purified 5.5-fold. Its molecular weight and Stoke's radius were estimated as 105,000 and 45.3 A, respectively. The Km for GTP was 0.98 microM. The temperature and pH optima for activity were 60-65 degrees C and 8.0-8.4, respectively. No divalent cation was required for the reaction. The pteridine product was 3'-triphosphate of 2-amino-4-hydroxy-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropteridine (dihydroneopterin triphosphate), identified by isolating its immediate derivative, 2',3'-cyclic phosphate of 2-amino-4-hydroxy-6-(D-erythro-1',2',3'-trihydroxypropyl)pteridine (neopterin cyclic phosphate). The radioactive product from [8-14C]GTP agreed with 14C-formate. Molar ratio of formate release to pteridine formation was 1.0.     

Production of monoclonal antibodies against human 6-pyruvoyl tetrahydropterin synthase and immunocytochemical localization of the enzyme.

Monoclonal antibodies were produced against human pituitary gland 6-pyruvoyl tetrahydropterin synthase, one of the key enzymes in the biosynthesis of tetrahydrobiopterin, by in vitro immunization with the antigen directly blotted from SDS-PAGE to polyvinylidene difluoride membranes. The antibodies produced show crossreactivity in the enzyme linked immunosorbent assay, not only with the human 6-pyruvoyl tetrahydropterin synthase but some also with the same enzyme isolated from salmon liver. 6-Pyruvoyl tetrahydropterin synthase was localized immuno-enzymatically in peripheral blood smears and in skin fibroblasts by the use of these monoclonal antibodies and the alkaline phosphatase monoclonal anti-alkaline phosphatase labeling technique.     

Tetrahydrobiopterin biosynthesis. Studies with specifically labeled (2H)NAD(P)H and 2H2O and of the enzymes involved

The biosynthesis of tetrahydrobiopterin from either dihydroneopterin triphosphate, sepiapterin, dihydrosepiapterin or dihydrobiopterin was investigated using extracts from human liver, dihydrofolate reductase and purified sepiapterin reductase from human liver and rat erythrocytes. The incorporation of hydrogen in tetrahydrobiopterin was studied in either 2H2O or in H2O using unlabeled NAD(P)H or (R)-(4-2H)NAD(P)H or (S)-(4-2H)NAD(P)H. Dihydrofolate reductase catalyzed the transfer of the pro-R hydrogen of NAD(P)H during the reduction of 7,8-dihydrobiopterin to tetrahydrobiopterin. Sepiapterin reductase catalyzed the transfer of the pro-S hydrogen of NADPH during the reduction of sepiapterin to 7,8-dihydrobiopterin. In the presence of partially purified human liver extracts one hydrogen from the solvent is introduced at position C(6) and the 4-pro-S hydrogen from NADPH is incorporated at each of the C(1') and C(2') position of BH4. Label from the solvent is also introduced into position C(3'). These results suggest that dihydrofolate reductase is not involved in the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate. They are consistent with the assumption of the occurrence of a 6-pyruvoyl-tetrahydropterin intermediate, which is proposed to be formed upon triphosphate elimination from dihyroneopterin triphosphate, and via an intramolecular redox reaction. Our results suggest that the reduction of 6-pyruvoyl-tetrahydropterin might be catalyzed by sepiapterin reductase.     

Biosynthesis of biopterin. Studies on the mechanism of 6-pyruvoyltetrahydropteridine synthase

[1'-3H]- and [2'-3H]dihydroneopterin triphosphate (NH2TP) were prepared enzymatically from [4-3H]- and [5-3H]glucose and converted to tetrahydrobiopterin (BH4) by an extract from bovine adrenal medulla. The formation of BH4 from both [1'-3H]- and [2'-3H]-NH2TP proceeds with virtually complete loss of the respective tritium label. The breaking of the CH-bond at C-1' is characterized by a kinetic isotope effect of 2.6 +/- 0.5. A smaller kinetic isotope effect of 1.5 +/- 0.2 was found for the breaking of the CH-bond at C-2'.     

Histidine 179 mutants of GTP cyclohydrolase I catalyze the formation of 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone triphosphate.

GTP cyclohydrolase I catalyzes the conversion of GTP to dihydroneopterin triphosphate. The replacement of histidine 179 by other amino acids affords mutant enzymes that do not catalyze the formation of dihydroneopterin triphosphate. However, some of these mutant proteins catalyze the conversion of GTP to 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5'-triphosphate as shown by multinuclear NMR analysis. The equilibrium constant for the reversible conversion of GTP to the ring-opened derivative is approximately 0.1. The wild-type enzyme converts the formylamino pyrimidine derivative to dihydroneopterin triphosphate; the rate is similar to that observed with GTP as substrate. The data support the conclusion that the formylamino pyrimidine derivative is an intermediate in the overall reaction catalyzed by GTP cyclohydrolase I.