Histidine 179 Mutants of GTP Cyclohydrolase I Catalyze the
Formation of
2-Amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone
Triphosphate*
Andreas
Bracher
,
Markus
Fischer
,
Wolfgang
Eisenreich
,
Harald
Ritz
,
Nicholas
Schramek
,
Peter
Boyle§,
Patrizia
Gentili§,
Robert
Huber¶,
Herbert
Nar¶,
Günter
Auerbach¶, and
Adelbert
Bacher
From the
Lehrstuhl für Organische Chemie und
Biochemie, Technische Universität München,
Lichtenbergstraße 4, D-85747 Garching, Germany, the
§ University Chemical Laboratory, Trinity College, Dublin 2, Ireland, and the ¶ Max-Planck-Institut für
Biochemie, Am Klopferspitz, D-82152 Martinsried, Germany
 |
ABSTRACT |
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.
 |
INTRODUCTION |
GTP cyclohydrolase I catalyzes the formation of dihydroneopterin
triphosphate from GTP via a mechanistically complex ring expansion. In
plants and micro-organisms, the enzyme product serves as the first
committed intermediate in the biosynthesis of tetrahydrofolate (1). In
animals, the enzyme product is converted to tetrahydrobiopterin, which
serves as cofactor for the biosynthesis of catecholamines and of nitric
oxide (2-4). Genetic defects of GTP cyclohydrolase I result in severe
neurological impairment (5-8).
GTP cyclohydrolase I of Escherichia coli is a 247-kDa
homodecamer (9, 10). The structure of the protein has been studied by
x-ray structure analysis at a resolution of 2.6 Å (11). The torus-shaped protein obeys D5 symmetry. Each of the 10 equivalent active sites is located at the interface of three adjacent subunits.
Brown, Shiota, and their co-workers (12-14) could show the reaction
sequence catalyzed by GTP cyclohydrolase I to involve the opening of
the imidazole ring of GTP (compound 1, Fig. 1) with release
of formate. Carbon atoms 1' and 2' of the ribose moiety of GTP are then
used to form the dihydropyrazine ring of dihydroneopterin triphosphate
(compound 5, Fig. 1). However, the mechanistic details of
the highly complex enzyme-catalyzed reactions are incompletely understood.
The catalytic activity of GTP cyclohydrolase is highly sensitive to the
replacement of amino acid residues at the active site cavity (11). The
amino acid residues Cys110, His112,
His113, Glu152, His179, and
Cys181 were shown to be indispensable for the formation of
dihydroneopterin triphosphate.1 This paper
describes the properties of mutant enzymes with replacement of
histidine 179.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Enzymes were purchased from Boehringer Mannheim
(Mannheim, Germany) and Sigma (Deisenhofen, Germany); GTP from Serva
(Heidelberg, Germany); [U-13C6]glucose was
purchased from Isotec (Miamisburg, Ohio),
15NH4Cl from ABCR (Karlsruhe, Germany), and
pteridine derivatives from Schircks Laboratories (Jona, Switzerland).
Sodium [13C]formate, phosphoryl chloride, trimethyl
phosphate, N,N-dimethylformamide, tri-n-butylamine, and pyrophosphoric acid were purchased
from Sigma-Aldrich. All other chemicals were reagent grade.
Enzyme Assays--
Assay mixtures contained 100 mM
Tris hydrochloride, pH 8.5, 100 mM KCl, 2.5 mM
EDTA, 1 mM GTP, and protein in a total volume of 450 µl.
The mixtures were incubated at 37 °C, and 100-µl aliquots were
retrieved at intervals. The formation of dihydroneopterin triphosphate
and
2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5'-phosphate was monitored as follows.
Assay of Dihydroneopterin Triphosphate--
Aliquots of enzyme
assay mixture were mixed with 30 µl of a solution containing 1%
iodine and 2% KI in 1 M HCl. After incubation for 30 min
at ambient temperature, excess iodine was reduced by addition of 0.11 M L-ascorbic acid (10 µl). After adjustment
to pH 8.5 by the addition of 1 M Tris-HCl, pH 8.5, a
solution (10 µl) containing 1.8 units of alkaline phosphatase, 0.2 µmol of MgCl2, and 0.26 µmol of ZnCl2 was
added. The mixture was incubated for 1 h at 37 °C. The reaction
was terminated by addition of trichloroacetic acid. Precipitate was
removed by centrifugation. Neopterin was determined by
HPLC2 as described earlier
(11).
Assay of
2-Amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone
5'-Triphosphate--
Aliquots (100 µl) of enzyme assay mixtures were
mixed with 100 µl of 1 M HCl. The mixtures were incubated
for 5 min at 90 °C. The pH was adjusted to 8.5 by the addition of 1 M NaOH. Butanedione in 1 M Tris-HCl, pH 8.5, was added to a final concentration of 0.5%. The mixtures were
incubated at 90 °C for 1 h. 6,7-Dimethylpterin was determined
by HPLC as described previously (16).
Preparation of
[8-13C]GTP--
[8-13C]Guanine was
prepared by the route used for
[7-15N,8-13C]guanine as described earlier
(17), except that unlabeled sodium nitrite was used. The crude material
thus obtained was purified by dissolving it in hot 2.5 M
HCl and treating with charcoal. Filtration of the hot solution gave a
yellow filtrate, which was neutralized to pH 7 with concentrated
NH4OH. Removal of some of the water on a rotary evaporator
and cooling the residue overnight at 5 °C gave
[8-13C]guanine as a yellowish powder. This was converted
(18) into [8-13C]guanosine, which was in turn converted
into the required [8-13C]GTP.
[8-13C]Guanosine (283 mg; 1 mmol), which had been dried
for 24 h over P2O5 under high vacuum, was
suspended in trimethyl phosphate (5 ml) under an atmosphere of dry
nitrogen contained in a flask fitted with a rubber septum and then
cooled in an ice bath (19). Freshly distilled phosphoryl chloride was
added from a syringe (0.285 ml; 3 mmol) and the mixture stirred at
0 °C. All solid material dissolved within 1 h. HPLC analysis
after 3 h (anion exchange column; phosphate gradient 0.02 M KH2PO4 to 0.5 M
KH2PO4) showed only one peak due to product and
no peak due to unreacted guanosine. To the cold solution was added cold
tri-n-butylamine (2 ml; 8.4 mmol), followed immediately by a
freshly prepared ice-cold solution of tri-n-butylamine
pyrophosphate, prepared from pyrophosphoric acid (1.780 g; 10 mmol) and
tri n-butylamine (4.765 ml; 20 mmol) in dimethylformamide
(10 ml) (20). After 1 min, the reaction mixture was quenched with cold
water (200 ml) and the pH adjusted to 9-10 with 1 M
aqueous potassium hydroxide. The mixture was extracted three times with
ether to remove free tributylamine, and all traces of ether were
removed from the aqueous layer using a rotary evaporator under vacuum
at room temperature. The aqueous solution was loaded onto a column of
Dowex-1 anion exchange resin in the chloride form (25 × 110 mm).
Distilled water (3.5 liters) was first passed through the column, which
was then developed with a sodium chloride gradient (0-0.2
M NaCl dissolved in 0.01 M HCl). Monitoring by
UV (254 nm) showed three well resolved peaks, the third and largest
being the desired GTP, eluted after about 7 liters of the eluting
gradient had been passed through. The GTP was separated from the buffer
salts by using a toluene-treated charcoal column. The type of charcoal
used for this purpose is critical, and some commercial charcoals are
not suitable. Riedel de Haën animal charcoal (knochenkohle
pulver, catalog number 18008) works satisfactorily. The charcoal column
(25 × 85 mm) was prepared in 1 M HCl and then washed
with water until the effluent was neutral. It was partially deactivated
by washing with a mixture of toluene/0.88 M
NH4OH/isopropanol/water (ratio 1:3:50:46), then with a mixture of EtOH/Water/NH4OH (2:2:1), and then with
distilled water. The column was stored in water and could be reused
repeatedly. The GTP-containing fractions from the Dowex column were
loaded onto the charcoal column, which was washed with distilled water (500 ml). Elution with 1 liter of a solution of ethanol/water/0.88 M NH4OH (50:45:5), followed by evaporation of
the eluate to dryness in a rotary evaporator under vacuum at room
temperature, gave a white powder, which was washed with ethanol and
diethyl ether and stored at
5 °C. Yield was 129 mg. HPLC analysis
of this product showed one main peak corresponding to authentic GTP
purchased from Sigma. Its UV spectra measured at pH 2, 7, and 13 were
identical with the corresponding UV spectra of authentic GTP.
1H, 13C, and 31P NMR spectra were
measured in buffer solution containing 20 mM Tris-HCl, pH
8.5, 2 mM EDTA, 2 mM dithiothreitol, 100 mM KCl, and 85% D2O and showed the product to
be pure GTP, uncontaminated with either guanosine di- or monophosphate,
although containing about 15% inorganic triphosphate. The
{1H}13C spectrum showed a single enriched
peak at
137.6 (C-8). The 1H spectrum included a doublet
at
8.03 (1JC-H = 216 Hz; H-8)
and a quartet at
5.82. (3JH-H = 6, 3JH-C = 4; H-1'). The
{1H}31P spectrum showed signals at
18.35 (t, J = 19.8 Hz; guanosine
-P),
7.51 (d,
J = 19.5 Hz; guanosine
-P), and
2.40 (d,
J = 21.1 Hz; guanosine
-P), as well as small signals
at
17.87 (t, J = 20.9 Hz; P-2 of inorganic
triphosphate) and
3.03 (d, J = 20.9 Hz; P-1 and P-3
of inorganic triphosphate). This material was used directly in the
biological experiments. 75% phosphoric acid was used as external reference.
Preparation of 13C,15N-Labeled
GTP--
[8,1',2',3',4',5'-13C6,7-15N]GTP
was prepared by published procedures (21) from
[U-13C5]ribose and
[7-15N,8-13C]guanine (17).
Preparation of
2-Amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone
5'-Triphosphate--
A mixture containing 8.5 mM potassium
phosphate, pH 7.0, 7 mM GTP (GDP-free), 85 mM
KCl, 2.1 mM EDTA, 4 mM dithiothreitol, and 40 mg of GTP cyclohydrolase I H179A mutant protein in a total volume of
4.7 ml was incubated for 6.5 h at 37 °C. The reaction was
terminated by the addition of 1 M HCl to a final pH of 4.7. Precipitate was removed by centrifugation. Ice-cold, doubly distilled water was added to a total volume of 50 ml. The solution was placed on
a column of DEAE-cellulose (DE52, Whatman, Maidstone, United Kingdom;
2 × 14.5 cm) that had been equilibrated with 30 mM
LiCl, pH 3.5, at 4 °C. The column was washed with 100 ml of 30 mM LiCl, pH 3.5, and was subsequently developed with a
linear gradient of 98-195 mM LiCl (total volume, 400 ml).
The effluent was monitored photometrically (280 nm). The yield (4.6 µmol, 13.5% based on GTP) was determined photometrically using an
absorbance coefficient of
273 nm, pH 7 = 14,300 M
1 cm
1. Fractions were
combined, and 0.5 ml of 0.25 mM ammonium bicarbonate was
added. The mixture was lyophilized. Dry methanol was added to the
residue. The insoluble lithium salt of
2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5'-triphosphate was collected by centrifugation. LiCl was removed by
washing with dry methanol. The residue was stored at
70 °C.
Preparation of
2,6-Diamino-5-formylamino-4(3H)-pyrimidinone--
2,6-Diamino-5-formylamino-4(3H)-pyrimidinone
was prepared according to the published synthesis by Pfleiderer
(22).
Site-directed Mutagenesis--
The point mutants of GTP
cyclohydrolase I were generated by a polymerase chain reaction strategy
(23). All mutated genes were sequenced with the dye terminator method
using an ABI Prism377 sequencer (Perkin-Elmer).
Cell Culture and Preparation of Crude
Extracts--
Escherichia coli strain M15[pREP4]
harboring the mutated pECHI expression plasmid was grown in LB medium
containing 150 mg of ampicillin and 22 mg of kanamycin per liter as
described (11). Cells were harvested by centrifugation. Wet cell mass
(200 mg) was suspended in 1 ml of 200 mM Tris-HCl, pH 8.0, containing 2.5 mM EDTA, 3 mM sodium azide, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 0.5 mg of lysozyme. The mixture was incubated for 90 min
at 37 °C. Cell lysis was completed by ultrasonic treatment. Cell
debris was removed by centrifugation.
Purification of GTP Cyclohydrolase I Mutant Protein
H179A--
Crude cell extract was dialyzed against 5 liters of buffer
A containing 10 mM potassium phosphate, pH 7.0, 2.5 mM EDTA, and 3 mM sodium azide. The dialysate
was applied to a column of DEAE-cellulose (DE52, Whatman; 4 × 11 cm) that had been equilibrated at 4 °C in the same buffer. The
column was washed with 300 ml of buffer A and developed with a linear
gradient of 10-150 mM potassium phosphate, pH 7.0. Fractions containing enzymatic activity (determined as formation of
2-amino5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5'-triphosphate) were combined and concentrated by ultracentrifugation. The protein was stored at 4 °C.
Estimation of Protein Concentration--
GTP cyclohydrolase I in
crude cell extracts was monitored by radial immune diffusion on agarose
plates containing 60 µl of polyclonal rabbit antiserum against
E. coli wild-type GTP cyclohydrolase I in 150 µl of 100 mM potassium phosphate, pH 7.0, containing 3 mM
sodium azide and 0.56% agarose (DNA-grade). Purified wild-type protein
was used as standard (21).
Total protein was determined by the method of Bradford (24, 25).
NMR Experiments--
One-dimensional and two-dimensional
1H and 13C NMR spectra were recorded at
17 °C in D2O using a Bruker DRX500 spectrometer. MLEV17
and GARP sequences were used for decoupling of 1H,
13C, and 15N. Experimental setup and data
processing were performed according to standard Bruker software (XWINNMR).
 |
RESULTS |
The hypothetical reaction mechanism of GTP cyclohydrolase I
proposed by Brown, Shiota, and their co-workers (12-14) (Fig.
1) implies the formation of at least two
ortho-diaminopyrimidine-type intermediates. Under
appropriate conditions, it appeared possible to convert compounds of
this type to highly fluorescent pteridines, thus facilitating their
detection with high sensitivity.
Our earlier site-directed mutagenesis study (11) had afforded a number
of GTP cyclohydrolase I mutants (point mutation of residues
Cys110, Cys181, Glu152,
His112, His113, and His179), which
were unable to form detectable amounts of dihydroneopterin triphosphate
from GTP. These apparently inactive mutants were incubated with GTP,
and the reaction mixtures were then boiled with HCl, neutralized, and
incubated with butanedione as described under "Experimental
Procedures" (Fig. 2). The mixtures were
analyzed by fluorescence-monitored HPLC.

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Fig. 2.
Assay of compound 2. A, 0.5 M HCl, 5 min at 90 °C; B, 0.5% butanedione
at pH 8.5 for 60 min at 90 °C.
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A fluorescent compound identified as 6,7-dimethylpterin (compound
6, Fig. 2) was observed in assays with cell extracts of
recombinant E. coli strains expressing mutant enzymes
carrying various amino acids instead of histidine 179, thus suggesting that a diaminopyrimidine-type product had indeed been formed. When the
HCl treatment was omitted, little or no 6,7-dimethylpterin was formed
in assays with these mutant proteins. This indicates that initially no
ortho-diaminopyrimidines were present. The rates of product
formation detected in these assay mixtures are summarized in Table
I. The highest formation rate of the
product detected as 6,7-dimethylpterin was observed with the H179S
mutant; mutants H179K and H179R did not catalyze any detectable
reaction at all. Nevertheless, the specific enzymatic activity for
formation of the unknown compound by mutant H179S was only 2.5% as
compared with the specific enzymatic activity for the formation of
dihydroneopterin triphosphate by wild-type GTP cyclohydrolase I. Mutant
H179A was selected for further studies on the basis of preliminary data on 6,7-dimethylpterin formation rate.
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Table I
Enzymatic activity as determined in crude lysates of expression strains
for GTP cyclohydrolase I mutants
Rates of product formation were obtained by linear regression analysis
of product concentrations versus time. GTP cyclohydrolase I
concentrations were determined by radial immune diffusion. ND, not
determined.
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Mutant protein H179A was purified from a recombinant E. coli
strain. Some properties are summarized in Table
II. The formation of the unknown product
from GTP by the H179A mutant is characterized by a value of
Km = 12 µM for GTP. For comparison, an apparent Km value of 0.85 µM GTP was
observed for the wild-type GTP cyclohydrolase I reaction yielding
dihydroneopterin triphosphate.
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Table II
Kinetic properties of wild-type GTP cyclohydrolase I and mutant H179A
All values were determined under standard assay conditions at pH 8.5 and 37 °C. The formation of 5 was monitored photometrically (330 nm)
as described (21). The conversion of 2 to GTP and vice versa
was monitored photometrically (250 nm) using a  of 8.97 × 10 3 µM 1 cm 1. Specific
activities were determined at substrate saturation. Wild-type GTP
cyclohydrolase I exhibited weak negative cooperativity. ND, not
determined.
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Whereas replacement of residue H179 affords mutant enzymes converting
GTP exclusively to the unknown product, the mutant proteins L134S,
Q151A, S135A, and S135C formed both the unknown compound and
dihydroneopterin triphosphate from GTP (Table I).
In order to determine the structure of the putative pyrimidine
intermediate produced by mutant H179A, we decided to use stable isotope
labeled GTP samples as substrates in order to increase the sensitivity
and selectivity of NMR detection. [8-13C]GTP was
synthesized and incubated with H179A mutant protein in phosphate buffer
at pH 7.0 in an NMR tube. The sample was kept in the NMR magnet at a
temperature of 37 °C, and 13C NMR spectra were recorded
at intervals (Fig. 3). A signal at 165.4 ppm increased at an approximately linear rate, and a signal at 164.9 was found to increase with apparently sigmoidal kinetics. After
incubation at 37 °C for 20 h, two small additional signals were
observed at 169.9 and 170.1 ppm. These initial data tentatively suggested the formation of four different formamide-type species from
the proffered GTP. Formation of significant amounts of
13C-labeled formate was not observed.

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Fig. 3.
Product formation from
[8-13C]GTP by GTP cyclohydrolase I mutant H179A in 10 mM potassium phosphate, pH 7.0, at 37 °C as monitored by
13C{1H} NMR spectroscopy. Spectra were
collected after 23 min (trace 1), 52 min (trace
2), 76 min (trace 3), 102 min (trace 4), 222 min (trace 5), and 1200 min (trace 6).
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The putative formamidopyrimidine-type compounds could be separated from
remaining substrate GTP by ion exchange chromatography. However, they
could not be separated from each other. In order to unequivocally
assign the structures of the four compounds formed, we prepared
[8,1',2',3',4',5'-13C6,7-15N]GTP
(Fig. 4A, compound
1a) by enzymatic synthesis. The compound was treated with
the H179A mutant protein, and the isolated mixture of four reaction
products was analyzed by two-dimensional NMR spectroscopy. Fig.
5 shows a section from a two-dimensional 1H13C HMQC-TOCSY spectrum and the corresponding
parts from one-dimensional 1H{13C} and
13C{1H} spectra. The one-dimensional
13C spectrum shows two pairs of doublets whose intensities
differ by a factor of about 10. These signals showed correlations to 1H doublet signals located in the range of 7.8 and 8.17 ppm. The chemical shift values of both 13C and
1H resonances are in line with the presence of four
different formamide-type molecular species. The doublet character of
the respective 13C and 1H signals can be
attributed to 15N coupling as demonstrated by comparison
with a 15N-decoupled spectrum where singlets are observed
(not shown). Therefore, all products observed carry the formyl group at
the 15N atom attached to position 5 of the pyrimidine
ring.

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Fig. 4.
Structural assignment of the isomeric product
mixture (B) obtained by the conversion of
[8,1',2',3',4',5'-13C6,7-15N]GTP
(A) with GTP cyclohydrolase I mutant H179A.
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Fig. 5.
Section from HMQC-TOCSY spectrum of the
product mixture obtained from
[8,1',2',3',4',5'-13C6,7-15N]GTP
by the mutant H179A.
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Chemical shift arguments suggest that the formamide C-N bond has
cis configuration in the pair of major products and
trans configuration in the pairs of minor products (26, 27).
The assignments are well in line with studies on the pyrimidine base aglycon, 2,6-diamino-5-formylamino-4(3H)-pyrimidinone, which
was also shown to display formamide cis/trans
isomerism. The 1H chemical shifts of the formyl groups in
this compound were closely similar to those of the products of GTP
cyclohydrolase I mutant H179A (Table
III). The ratio of the cis and
trans species in the equilibrium mixture of
2,6-diamino-5-formylamino-4(3H)-pyrimidinone were also in
close similarity with that in the product mixture generated by the
H179A mutant enzyme.
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Table III
NMR data of the compounds discussed under Results
Spectra were measured in D2O at pH 7.0. ND, not determined.
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Fig. 6 shows an expansion comprising the
carbohydrate signals of a two-dimensional HMQC experiment obtained with
the same sample as in Fig. 5. Cross-peaks correlate 13C
atoms with directly bonded 1H atoms. In conjunction with
HMQC-TOCSY and 13C INADEQUATE spectra (not shown), the
signals in this spectrum can be assigned unequivocally to an anomeric
mixture of N-ribofuranosides for the major product pair
(Table III). Therefore, the products of the conversion of GTP with GTP
cyclohydrolase I mutant H179A have the same connectivities but differ
with regard to the configuration at the anomeric center and at the
formamide bond (Fig. 4B). Specifically, compounds
2a and 2c carry the ribofuranose triphosphate in
-glycosidic linkage, and compounds 2b and 2d are
-glycosides.

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Fig. 6.
Section from HMQC spectrum of the product
mixture obtained from
[8,1',2',3',4',5'-13C6,7-15N]GTP
by mutant H179A.
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The data in Fig. 3 indicate that the
cis isomer is
formed at a linear initial rate, whereas the
cis isomer
is formed with sigmoid kinetics. This suggests that the
cis isomer is formed in the enzyme catalyzed reaction and is
subsequently converted to the other products by nonenzymatic reactions.
Specifically, spontaneous (i.e. nonenzymatic) anomerization
of the glycosidic bond affords the
cis isomer.
Similarly, the formamide cis/trans isomerization
appears to be a nonenzymatic reaction.
Quantitative analysis of the NMR data at equilibrium shows that the
equilibrium between
- and
-glycosides is characterized by an
equilibrium constant close to 1. Moreover, it can be seen that the
equilibrium ratio between trans and cis isomer is
approximately 1:10. This is well in line with experimental and
theoretical data indicating that the cis isomer of
monosubstituted formamides is 1.4-1.6 kcal/mol more stable than the
trans isomer (15, 28, 29).
The enzyme reaction catalyzed by the H179A protein can be monitored
photometrically. Fig. 7A shows
the reaction from GTP to the formylaminopyrimidine product. The UV
spectra of the isomeric mixture of
2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone triphosphates are shown in Fig. 7C for comparison. Fig.
7B shows the reverse reaction using the mixture of four
stereoisomers as substrate. The rate of the reverse reaction was
approximately two times higher than that of the forward reaction. The
reverse reaction slowed down significantly after turnover of about 40% of the formamides present. This is caused by exhaustion of the
isomer fraction. The equilibrium constant for the reversible conversion
between GTP and the
cis isomer is approximately 0.1.

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Fig. 7.
Ultraviolet spectra recorded during the
conversion of GTP with mutant H179A (A) and during the
conversion of the isomeric mixture of
2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone
triphosphate (compound 3) with mutant H179A (B) at
30 °C. Reaction mixture A contained 83 µM GTP, 83 µg/ml mutant H179A, 10 mM Tris-HCl, pH 8.5, 10 mM KCl, and 0.25 mM EDTA. Reaction mixture B
contained a total amount of 99 µM compounds
2a-2d and 208 µg/ml mutant H179A in the same
buffer as above. The insets show difference spectra with the
initial spectrum as reference. For comparison, the UV spectra of
compounds 2a-2d (equilibrium mixture) and of GTP at pH 7.0 are shown (C).
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Following on the treatment of GTP with Fenton's reagent
(Fe2+ and mercaptoethanol in the presence of molecular
oxygen), Shiota and co-workers (13, 14) isolated a compound tentatively
identified as compound 2 (Fig. 1). Using the published
procedure, we isolated a product mixture that had the same UV spectrum
and 1H NMR signature as the product mixture obtained from
treatment of GTP with the H179A protein (data not shown).
Shiota and co-workers (13, 14) had already shown that the product
obtained with Fenton's reagent from GTP could be converted to
dihydroneopterin triphosphate by GTP cyclohydrolase I in cell extracts
of Lactobacillus plantarum. We confirmed that the formamide mixture obtained by treatment of GTP with the H179A mutant protein could be converted to dihydroneopterin triphosphate by wild-type E. coli GTP cyclohydrolase I. Similarly, we could show that
the product obtained by treatment of GTP with Fenton's reagent
according to Shiota's procedure could be converted to dihydroneopterin
triphosphate. The rate of product formation (as determined by the
increase of absorbance at 330 nm) was similar to the rate of the
reaction with the natural substrate GTP (Table II). NMR experiments
with [formyl-13C]2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone triphosphate obtained enzymatically from [8-13C]GTP (data
not shown) confirmed that the
cis isomer is
preferentially consumed when the mixture is incubated with wild-type
enzyme. In summary, the kinetic competency of compound 2a
suggests that it is an intermediate in the GTP cyclohydrolase I
reaction trajectory.
Although unknown at present, mutants may be discovered that, while
unable to utilize GTP as a substrate, are able to convert the product
mixture of mutant His179 to dihydroneopterin triphosphate.
The mutants C110S, H112S, H113S, and C181S are unable to generate
dihydroneopterin triphosphate from GTP.1 None of these
mutants, however, led to any detectable dihydroneopterin triphosphate
when incubated with the H179A product mixture.
 |
DISCUSSION |
Aqueous solutions of
2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone
triphosphate obtained by the catalytic action of the H179A mutant of
GTP cyclohydrolase I contain a mixture of four stereoisomers
2a-2d. The isomerization reactions between the
components probably occur nonenzymatically. Mutarotation of
N-glycosides is a proton-catalyzed reaction. From the series of 13C NMR spectra collected during conversion of
[8-13C]GTP with mutant H179A a reaction rate for
mutarotation of 0.01 min
1 at pH 7.0 and at a temperature
of 37 °C can be estimated. Kenne et al. (27) determined a
reaction rate of 0.02 s
1 (at 30 °C) for the formamide
isomerization of the model compound methyl
4,6-dideoxy-4-formylamino-
-D-mannopyranoside.
The
cis isomer of
2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone
triphosphate appears to be an intermediate in the transformation of GTP
to dihydroneopterin triphosphate catalyzed by GTP cyclohydrolase I on
the basis of the following arguments. (i) The
cis isomer
is the primary product of the GTP cyclohydrolase I mutant H179A,
whereas the signal for the
cis isomer is characterized
by sigmoidal kinetics of formation. (The concentration of the
trans isomers comprising the minor products of mutant H179A
is too small for any interpretation.) (ii) The
cis
isomer comprising the major portion of the
-anomeric isomers is
preferentially consumed by the H179A mutant during the formation of
GTP. (iii) The
cis isomer is preferentially converted to dihydroneopterin triphosphate by wild-type enzyme. (iv) The rate of
formation of dihydroneopterin triphosphate (compound 5) from
the isomeric mixture by the wild-type enzyme is similar to its rate of
formation from GTP. However, we cannot rule out the possibility that
the
cis isomer is in equilibrium with an undetected intermediate of the reaction sequence without being itself a proper intermediate. It remains unknown whether formamide
cis/trans isomerization plays a mechanistic role
in the GTP cyclohydrolase I reaction sequence. These considerations are
summarized in Fig. 8.

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|
Fig. 8.
Proposed reaction scheme for the formation of
the formamide isomeric mixture by enzyme-catalyzed and by nonenzymatic
reactions. WT, wild-type enzyme.
|
|
The conversion of GTP to the formamidopyrimidine nucleotide
2a by H179 mutant proteins is reversible. The equilibrium constant has a value of about 0.1 in aqueous solution at neutral pH.
The reversible interconversion of a formamide-type intermediate and GTP
reaction is reminiscent of the chemical synthesis of guanine from
2,5,6-triamino-4(3H)-pyrimidinone and formic acid. The
latter reaction, however, requires high temperature and the absence of water. The direct formation of a purine nucleoside by ring closure to
form the imidazole ring has not been reported to the best of our knowledge.
On the basis of x-ray structure data obtained with the wild-type
enzyme, we had proposed earlier that residue H179 is involved in the
opening of the imidazole ring of GTP (11). Specifically, it appeared
likely that the reaction could be initiated by protonation of N7 of
substrate GTP by the amino acid residue. Whereas some mutants with
replacement of His179 can still catalyze the ring opening
reaction, the maximum velocity observed with the H179S mutant is about
3% as compared with the rate of formation of dihydroneopterin
triphosphate by the wild-type. Moreover, preliminary stopped flow
experiments suggest that the opening of the imidazole ring by the
wild-type enzyme is much faster than the overall reaction. Thus, it
appears that the cleavage of the bond between N7 and C8 of GTP is
slowed down by more than 1 order of magnitude if His179 is
replaced. The slow residual reaction could be enabled by protonation of
N7 of the substrate by another amino acid in the neighborhood.
The data also show that His179 is essential for the removal
of formate from compound 2 by cleavage of the formamide
bond. This would imply that the imidazole moiety of His179
catalyzes two mechanistically important steps at a
biosynthetically equivalent nitrogen atom (viz. N-7 of
GTP and N-5 of compound 2).
The formamidopyrimidine compound 2 is also observed in
enzyme assays conducted with mutants of Gln151,
Leu134, and Ser135. Glutamine 151 has been
proposed to form a hydrogen bond with His179 in the
wild-type enzyme. This could be relevant for optimum positioning of
His179 for the hydrolysis of the formamide bond.
We showed recently1 that several mutants of the amino acids
Cys110, His112, His113, and
Cys181 are able to bind GTP in an apparently normal
fashion, but do not catalyze any reaction. In the present paper we have
shown further that these same mutants are unable to convert compound 2 to dihydroneopterin triphosphate. It appears, therefore, that their inability to produce dihydroneopterin triphosphate from GTP
is not due solely to their inability to catalyze the initial stages of
the reaction.
 |
ACKNOWLEDGEMENT |
We thank A. Werner for expert help with the
preparation of this manuscript.
 |
FOOTNOTES |
*
This work was supported by the Deutsche
Forschungsgemeinschaft, by European Community Grants ERB CHRX CT93-0243
and ERB FMRX-CT98-0204, and by the Fonds der Chemischen Industrie.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Lehrstuhl
für Organische Chemie und Biochemie, TU München,
Lichtenbergstraße 4, D-85747 Garching, Germany. Tel.: 49-89-289-13360;
Fax: 49-89-289-13363; E-mail: adelbert.bacher{at}ch.tum.de or
bacher{at}bionmr.org.chemie.tu-muenchen.de.
1
G. Auerbach, A. Bracher, H. Nar, M. Fischer, C. Hösl, R. Huber, and A. Bacher, manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
HPLC, high
performance liquid chromatography;
HMQC, heteronuclear multiple quantum
coherence;
TOCSY, totally correlated spectroscopy.
 |
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