Ring Opening Is Not Rate-limiting in the GTP Cyclohydrolase I Reaction*

Nicholas Schramek, Andreas BracherDagger, and Adelbert Bacher§

From the Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany

Received for publication, June 6, 2000, and in revised form, October 19, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 k1 for the reversible opening of the imidazole ring of GTP was 0.9 s-1, the rate constant k3 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 k4 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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GTP cyclohydrolase I catalyzes a ring expansion affording dihydroneopterin triphosphate and formate from GTP (Fig. 1) (1, 2). The enzyme product is the first intermediate in the biosynthesis of folate coenzymes in plants and many microorganisms (3). In animals, the product of GTP cyclohydrolase I is the first committed intermediate in the biosynthesis of tetrahydrobiopterin, which is required as a cofactor for the biosynthesis of catecholamines and nitric oxide (4-6).



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Fig. 1.   Hypothetical reaction mechanism of GTP cyclohydrolase I.

The molecular structure of GTP cyclohydrolase I has been studied in considerable detail. The enzyme from Escherichia coli is a D5 symmetric decamer of 250 kDa from which a three-dimensional structure has been solved by x-ray crystallography at a resolution of 2.4 Å (7). An essential zinc ion at the active site was discovered only recently by crystallographic studies with human GTP cyclohydrolase I (8).

The reaction mechanism of GTP cyclohydrolase is complex. 2-Amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate (Compound 4, Fig. 1) has been established as an early reaction intermediate by work with histidine 179 (H179A) mutants of the E. coli enzyme (9). The ring opening reaction is reversible with an equilibrium constant of 0.1. The formation of the 5-aminopyrimidine derivative (Compound 5) by hydrolysis of the formamide intermediate (Compound 4) has been suggested by work with arginine 185 (R185A) mutants.1 Experimental evidence for a subsequent Amadori rearrangement preceding the formation of the dihydropterin system has been obtained recently by deuterium tracer experiments monitored by NMR spectroscopy, which showed that a hydrogen atom is incorporated into the pro-7R position of dihydroneopterin triphosphate from solvent water (10).

It should be noted that Fig. 1 represents at best a simplified model of the reaction sequence that involves a complex series of intermediates and transition states. This paper reports an attempt to dissect this complex reaction trajectory by single turnover quenched flow experiments.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- [8-14C]GTP was purchased form Moravek Biochemicals Inc. (Brea, Canada). Recombinant GTP cyclohydrolase I of E. coli (10) and GTP cyclohydrolase I mutant protein H179A of E. coli (9) were prepared as described earlier.

Enzyme Assay-- Assay mixtures contained 50 mM Tris/HCl, pH 8.5, 100 mM KCl, 0.4 mM GTP, and protein in a total volume of 1 ml. The mixtures were incubated at 37 °C, and 100-µl aliquots were retrieved at intervals. The formation of 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone 5'-triphosphate (Compound 4) (273 nm, epsilon 273 nm = 14,300 M-1 cm-1 (9)) and the concentration of GTP were monitored photometrically by HPLC2 as described later.

Determination of Zinc-- To a solution of enzyme (3 mg/ml) in buffer, 50 mM Tris/HCl, pH 8.0, hydrochloric acid was added to a final acid concentration of 1 M. This mixture was heated to 96 °C for 2 h. Zinc was then determined by atomic absorption spectrometry using a Unicam 919 spectrometer (Unicam, Cambridge, United Kingdom).

Quenched Flow Experiments-- Quenched flow experiments were performed with an SFM4/QS apparatus from Bio-Logic (Claix, France) equipped with a linear array of mixers, four independent syringes, and a computer-controlled valve. The apparatus was thermostated at 30 °C. A delay loop of 230 µl of nominal volume was used. Syringe 1 contained a reaction buffer consisting of 50 mM Tris/HCl, pH 8.5, 100 mM KCl, and 0.002% sodium azide. Syringe 2 contained 125 µM [8-14C]GTP (320 µCi mmol-1) in reaction buffer. Syringe 3 contained 34 µM GTP-cyclohydrolase I (equivalent to 340 µM active sites) in reaction buffer. At the start of each experiment, the delay loop was washed with reaction buffer from syringe 1 and was then loaded with substrate solution from syringe 2 and with enzyme solution from syringe 3. The solutions were mixed at a ratio of 1:1 (v/v) and at a flow rate of 4 ml/s. After an appropriate delay, the reaction mixture in the delay loop was quenched by mixing with 0.2 M trichloroacetic acid from syringe 4 at a ratio of 1:1 (v/v) and a flow rate of 4 ml min-1. The effluent was collected. Samples were centrifuged to remove protein and were frozen at -70 °C until further analysis.

HPLC Analysis of the Reaction Mixtures-- Aliquots of 100 µl were injected into a reversed phase column (4.6 × 250 mm, Hypersil RP18, 5 µm, Schambeck, Bad Honnef, Germany). The column was developed with isopropyl alcohol, triethylamine, 85% phosphoric acid, water (8:10:3:979 (v/v), pH 7). Separations were performed at room temperature with a flow rate of 1 ml min-1. The effluent was monitored by a continuous flow scintillation counter (Berthold LB 503, Wildbad, Germany) and a diode array photometer (J&M, Aalen, Germany). The concentration of GTP was determined photometrically (252 nm, epsilon 252 nm = 13,700 M-1 cm-1 (11)) as well as by scintillation counting; 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate (Compound 4) and formate were measured by scintillation counting, and dihydroneopterin triphosphate was monitored photometrically (330 nm, epsilon 330 nm = 6,300 M-1 cm-1 (12)).

Calculations-- All calculations were run on an Intel PIII-600 personal computer. Rate constants were calculated by the program Scientist 2.02 (MicroMath, Inc., Salt Lake City, UT).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GTP cyclohydrolase I is an inherently slow enzyme. The turnover rate under steady state conditions is 0.05 s-1/subunit, similar to that of G proteins that serve regulatory functions rather than metabolic functions. This low rate is not unusual for an enzyme involved in the biosynthesis of a minor metabolite, and it provides favorable conditions for analysis by single turnover quenched flow experiments.

Quenched flow experiments were performed as described under "Experimental Procedures" using [8-14C]GTP as substrate. A 3-fold excess of enzyme subunits over substrate was used to minimize the effects of the negative cooperativity of GTP cyclohydrolase I that had been reported earlier (9).

The enzyme-catalyzed reaction was allowed to proceed for periods of 0.125-150 s. The reaction was then quenched with trichloroacetic acid, and the reaction mixture was analyzed by HPLC and monitored by multiwavelength photometry and scintillation counting. Compound 4 and formate were assessed on the basis of the radioactivity measurements, dihydroneopterin triphosphate was assessed on the basis of absorbance at 330 nm (Fig. 2), and GTP was assessed on the basis of photometry at 254 nm as well as scintillation counting. Other intermediates could not be assessed quantitatively because of overlapping HPLC peaks.



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Fig. 2.   HPLC detection of dihydroneopterin triphosphate in samples from quenched flow experiments. For experimental conditions see "Experimental Procedures."

Data from a typical quenched flow experiment are summarized in Fig. 3 and afford several preliminary conclusions: (i) the consumption of GTP is biphasic by which a rapid decrease to a level around 20% is followed by a very slow further decrease; (ii) the formation of formate proceeds relatively rapidly and is virtually complete after 15 s; (iii) the formation of dihydroneopterin triphosphate is substantially less rapid and reaches its 50% value after about 30 s; and (iv) in line with the expectation, dihydroneopterin triphosphate and formate are formed in stoichiometric amounts, and their final concentrations are equivalent to consumed GTP.



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Fig. 3.   Single turnover kinetics of GTP cyclohydrolase I. black-square, GTP, open circle , 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone triphosphate (Compound 4); , formate; black-down-triangle , dihydroneopterin triphosphate. The curves are exponential approximations.

The incomplete consumption of GTP, at least during a period of more than 100 s, is not easily explained in light of the 3-fold excess of active sites over the profferred substrate. From the crystallographic studies, it is known that GTP cyclohydrolase I of E. coli can easily lose zinc (8). In fact, the metal ion had not been found in the initial crystallographic studies (7) because substantially zinc-depleted protein samples had been inadvertently used, and a crystal structure of the E. coli enzyme that bound zinc had been obtained only recently (8). Unfortunately, the reconstitution of a partially zinc-depleted enzyme has not been possible because the enzyme precipitates upon the addition of zinc salts even at low concentrations.

We assume tentatively that GTP can be trapped in active sites devoid of zinc and can thus escape catalytic conversion for long periods because the metal ion is absolutely required for catalytic activity (it is known from the crystallographic studies that the zinc-depleted enzyme can bind GTP tightly). In typical enzyme preparations, the zinc content was ~0.8 zinc ion/subunit as estimated from atomic absorption spectrometry. These data are in agreement with the fraction of GTP (~15%) remaining after a reaction time of 3 min in a substoichiometric quenched flow experiment. Slow reequilibration of GTP trapped in metal-depleted active sites with the bulk solvent could then explain the continuing slow consumption of GTP extending over many minutes.

The enzyme-catalyzed formation of Compound 4 from GTP is a reversible reaction (9). Using the H179A mutant of GTP cyclohydrolase I that can convert GTP to Compound 4 but not beyond that intermediate, a value of 0.1 has been reported earlier for the equilibrium constant at pH 7. In the present study, we found a value of 0.14 at pH 8.5 and 30 °C by steady state analysis of the reaction catalyzed by the H179A mutant of GTP cyclohydrolase I. The release of formate by the hydrolysis of Compound 4 should be essentially irreversible on entropic grounds. Because formate is a final product of the GTP cyclohydrolase reaction, it is possible to limit the analysis to the initial steps of single turnover experiments that are conducive to the formation of formate from GTP as summarized in the reaction Scheme 1.



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Scheme 1.  

Scheme 1 can be described by the following set of differential equations (Model I):
<FR><NU>d[<B><UP>1</UP></B>]</NU><DE>dt</DE></FR>=−k<SUB>1</SUB>·[<B><UP>1</UP></B>]+<FR><NU>k<SUB>1</SUB></NU><DE>K</DE></FR>·[<B><UP>4</UP></B>] (Eq. 1)

<FR><NU>d[<B><UP>4</UP></B>]</NU><DE>dt</DE></FR>=k<SUB>1</SUB>·[<B><UP>1</UP></B>]−<FR><NU>k<SUB>1</SUB></NU><DE>K</DE></FR>·[<B><UP>4</UP></B>]−k<SUB>3</SUB>·[<B><UP>4</UP></B>] (Eq. 2)

<FR><NU>d[<B><UP>5</UP></B>]</NU><DE>dt</DE></FR>=k<SUB>3</SUB>·[<B><UP>4</UP></B>] (Eq. 3)

K=<FR><NU>k<SUB>1</SUB></NU><DE>k<SUB>2</SUB></DE></FR>=<FR><NU>[<B><UP>4</UP></B>]<SUB><UP>eq</UP></SUB></NU><DE>[<B><UP>1</UP></B>]<SUB><UP>eq</UP></SUB></DE></FR> (Eq. 4)
The program Scientist 2.02 (MicroMath, Inc.) was used to fit that set of equations to the experimental data (Fig. 4). The GTP concentration data were corrected for the fraction of GTP that remained intact even after a period of more than 100 s. The best fit was obtained with the rate constants summarized in Table I.



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Fig. 4.   Formation of formate by GTP cyclohydrolase I. Experimental values for concentrations are indicated as follows: black-square, GTP; , formate; and open circle , 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone triphosphate (Compound 4). The optimized concentration profiles according to kinetic Model I are represented as lines.


                              
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Table I
Rate constants of the GTP cyclohydrolase I reaction

The concentration of Compound 4 reaches a maximum after about 1 s, and the ratio of Compound 4 and GTP approaches a plateau value of 0.175 ± 0.025 (Fig. 5). This value corresponds within the limits of experimental accuracy to the equilibrium constant of 0.14 observed at pH 8.5 and 30 °C under steady state conditions with the H179A mutant (see above).



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Fig. 5.   Ratio of Compound 4 (2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone triphosphate) to GTP. The curve was calculated with values from Table I (Model I).

The final part of the reaction sequence involves the formation of the dihydropteridine ring system from Compound 5 under elimination of a water molecule. The newly formed double bond is conjugated to the pi -electron system of the pyrimidinone ring. The reaction is therefore expected to be exergonic and practically irreversible. Because it is obvious from Fig. 1 that Compound 5 is formed from Compound 4 at the same rate as formate, a simplified kinetic scheme for the formation of dihydroneopterin triphosphate from GTP can be rendered as Scheme 2.



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Scheme 2.  

Scheme 2 is described by the following set of differential equations (Model II):
<FR><NU>d[<B><UP>1</UP></B>]</NU><DE>dt</DE></FR>=−k<SUB>1</SUB>·[<B><UP>1</UP></B>]+<FR><NU>k<SUB>1</SUB></NU><DE>K</DE></FR>·[<B><UP>4</UP></B>] (Eq. 5)

<FR><NU>d[<B><UP>4</UP></B>]</NU><DE>dt</DE></FR>=k<SUB>1</SUB>·[<B><UP>1</UP></B>]−<FR><NU>k<SUB>1</SUB></NU><DE>K</DE></FR>·[<B><UP>4</UP></B>]−k<SUB>3</SUB>·[<B><UP>4</UP></B>] (Eq. 6)

<FR><NU>d[<B><UP>5</UP></B>]</NU><DE>dt</DE></FR>=k<SUB>3</SUB>·[<B><UP>4</UP></B>]−k<SUB>4</SUB>·[<B><UP>5</UP></B>] (Eq. 7)

<FR><NU>d[formate]</NU><DE>dt</DE></FR>=k<SUB>3</SUB> · [4] (Eq. 8)

<FR><NU>d[10]</NU><DE>dt</DE></FR>=k<SUB>4</SUB> · [5] (Eq. 9)

K=<FR><NU>k<SUB>1</SUB></NU><DE>k<SUB>2</SUB></DE></FR>=<FR><NU>[4]<SUB><UP>eq</UP></SUB></NU><DE>[1]<SUB><UP>eq</UP></SUB></DE></FR> (Eq. 10)
The fitting of these equations to the experimental data was again performed with the program Scientist 2.02 (Fig. 6) using k1, k2, and k3 values from the previous simulation as initial parameters. The rate constants for the best fit are shown in Table I.



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Fig. 6.   Numerical optimization of reaction Model II for dihydroneopterin triphosphate formation by GTP cyclohydrolase I. The following symbols were used for experimentally determined concentrations: black-square, GTP; open circle , 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone triphosphate; , formate; black-triangle, dihydroneopterin triphosphate. Lines represent concentrations according to kinetic Model II. The same data are shown at different scaling in A and B.

The formation of dihydroneopterin triphosphate from Compound 5 proceeds with a rate constant of 0.03 s-1. Similar values of k1, k2, and k3 were obtained in the simulation according to Model I and Model II (Table I). It follows that the Amadori rearrangement or the subsequent ring closure reaction is limiting the rate of the overall reaction. However, it should be noted that the rate constant k4 probably does not describe a single reaction step.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GTP cyclohydrolase I catalyzes a complex reaction sequence involving the cleavage of two different CN bonds and a rearrangement of the carbohydrate side chain followed by the formation of the pyrazine ring of the product, dihydroneopterin triphosphate. Moreover, a conformational reorientation of the reactant appears to be required in the second part of the reaction trajectory.

Recent biochemical and crystallographic studies have shown that a zinc ion bound to amino acid residues Cys-110, Cys-181, and His-113 is absolutely required for the hydrolytic opening of the imidazole ring of GTP (8). GTP in complex with the C110S mutant protein that is unable to bind zinc remained stable during a period of several months.3 It is at present unknown whether the zinc ion is also involved in the Amadori rearrangement of the carbohydrate side chain.

Saturation mutagenesis of the active site amino acid residues has also shown that histidine 179 is absolutely required for the cleavage of the formamide bond of Compound 4 (9).

This study reports the concentrations of intermediates subsequent to acid quenching of the reaction mixture. The possibility must be considered that certain intermediates could undergo secondary chemical reactions as a consequence of the acid treatment. Specifically, the hypothetical GTP hydrate, Compound 2, could be converted to GTP or Compound 4 (Compound 3 is significantly less stable) by the acid treatment. Because ring opening is rapid by comparison with the overall enzyme reaction, this uncertainty appears tolerable.

The kinetic scheme used for the interpretation of the experimental data represents a rigorous simplification. It can be estimated that about two dozen rate constants would be required for a comprehensive description including all reaction steps. There can be no doubt that the experimental system is substantially underdetermined. However, it is also likely that several reaction steps, in particular the numerous proton transfer reactions required, are rapid by comparison with the reactions described by k1 to k4 and would therefore not influence the overall catalytic rate of the enzyme to a significant degree.

The interpretation of the data substantially benefits from the fact that one of the final products, formate, is released early in the reaction sequence. Therefore, it was justified to simulate the conversion of GTP to formate rather than to dihydroneopterin triphosphate. This simplified approach afforded starting values for the rate constants k1-k3. The use of that set of parameters resulted in the rapid convergence of the subsequent simulation involving all experimental data (including the formation of dihydroneopterin triphosphate) and the kinetic constants k1-k4. Both numerical analyses gave similar values for k1-k3 within the experimental limits. It should also be noted that the ratio of k1/k3 is numerically similar to the estimated equilibrium constant for the reversible conversion of GTP to Compound 4 within the limits of experimental accuracy. The internal consistency of these data supports the validity of the simplified kinetic scheme used for data interpretation.

The carbohydrate side chain isomerization catalyzed by GTP cyclohydrolase I is similar to the Amadori rearrangement catalyzed by phosphoribosyl anthranilate isomerase in the tryptophan biosynthetic pathway (Fig. 7). The turnover number of phosphoribosyl anthranilate isomerase (bifunctional enzyme from E. coli) is 40 s-1 (13). By comparison, the rate constant for the conversion of Compound 5 to dihydroneopterin triphosphate is 0.03 s-1. These reaction rates differ by three orders of magnitude. The Amadori rearrangement and/or ring closure reaction step is rate-limiting in the overall GTP cyclohydrolase I reaction sequence.



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Fig. 7.   Reaction catalyzed by phosphoribosyl anthranilate isomerase and indolyl glycerol phosphate synthase.

GTP cyclohydrolase I catalyzes the first and possibly rate-determining step in the biosynthesis of trace metabolites (tetrahydrofolate in bacteria and plants, tetrahydrobiopterin in animals). The very low overall rate of the enzyme is similar to those of other enzymes involved in the biosynthesis of heterocyclic vitamins and coenzymes (Table II). These low reaction rates are likely to reflect the metabolic needs of the respective organisms rather than a high activation barrier of the respective reactions per se. GTP cyclohydrolase I is also likely to have evolved for a low reaction rate. However, it is surprising that the terminal part of the reaction sequence rather than one of the initial steps is actually rate-limiting.


                              
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Table II
Turnover rates of enzymes involved in the biosynthesis of heterocyclic vitamins and coenzymes



    ACKNOWLEDGEMENT

We thank A. Werner for expert help with the preparation of this manuscript.


    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and the Hans-Fischer-Gesellschaft.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.

Dagger Present address: European Molecular Biology Laboratory, 6 rue Jules Horowitz, F-38000 Grenoble, France.

§ To whom correspondence should be addressed. Tel.: 49-89-289-13360; Fax: 49-89-289-13363; E-mail: adelbert.bacher@ch.tum.de.

Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M004912200

1 A. Bracher, unpublished data.

3 C. Hösl, unpublished data.


    ABBREVIATIONS

The abbreviation used is: HPLC, high pressure liquid chromatography..


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Burg, A. W., and Brown, G. M. (1968) J. Biol. Chem. 243, 2349-2358[Abstract/Free Full Text]
2. Shiota, T., Palumbo, M. P., and Tsai, L. (1967) J. Biol. Chem. 242, 1961-1969[Abstract/Free Full Text]
3. Brown, G. M., and Williamson, J. M. (1987) in Escherichia coli and Salmonella typhimurium (Neidhardt, F. C., ed), Vol. 1 , pp. 521-538, American Society for Microbiology, Washington, D. C.
4. Tayeh, M. A., and Marletta, M. A. (1989) J. Biol. Chem. 264, 19654-19658[Abstract/Free Full Text]
5. Nichol, C. A., Smith, G. K., and Duch, D. S. (1985) Annu. Rev. Biochem. 54, 729-764[CrossRef][Medline] [Order article via Infotrieve]
6. Kwon, N. S., Nathan, C. F., and Stuehr, D. J. (1989) J. Biol. Chem. 264, 20496-20501[Abstract/Free Full Text]
7. Nar, H., Huber, R., Meining, W., Schmid, C., Weinkauf, S., and Bacher, A. (1995) Structure 3, 459-466[Medline] [Order article via Infotrieve]
8. Auerbach, G., Herrmann, A., Bracher, A., Bader, G., Gütlich, M., Fischer, M., Neukamm, M., Garrido-Franco, M., Richardson, J., Nar, H., Huber, R., and Bacher, A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13567-13572[Abstract/Free Full Text]
9. Bracher, A., Fischer, M., Eisenreich, W., Ritz, H., Schramek, N., Boyle, P., Gentili, P., Huber, R., Nar, H., Auerbach, G., and Bacher, A. (1999) J. Biol. Chem. 274, 16727-16735[Abstract/Free Full Text]
10. Bracher, A., Eisenreich, W., Schramek, N., Ritz, H., Götze, E., Herrmann, A., Gütlich, M., and Bacher, A. (1998) J. Biol. Chem. 273, 28132-28141[Abstract/Free Full Text]
11. Dawson, R. C. M., Elliot, D. C., Elliot, W. H., and Jones, K. M. (1986) Data for Biochemical Research , 3rd Ed. , p. 109, Oxford University Press, Oxford
12. Pfleiderer, W. (1985) in Folates and Pterins (Blakely, R. L. , and Benkovic, S. J., eds), Vol. 2 , pp. 43-114, John Wiley & Son, New York
13. Hommel, U., Eberhard, M., and Kirschner, K. (1995) Biochemistry 34, 5429-5439[Medline] [Order article via Infotrieve]


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