Biosynthesis of Guanosine 5’-Phosphate

I. XANTHOSINE F-PHOSPHATE AS AN INTERMEDIATE*

ULF LAGERKVIST

From the Chemical Department, Karolinska Institutet, Stockholm, Sweden

(Received for publication, January 14, 1958)

The work of Greenberg (1) and Buchanan et al. (2) has es- tablished inosine 5’-phosphate as a key intermediate in the biosynthesis of hypoxanthine in pigeon liver. The recent indication of the enzymic synthesis of adenosine 5’-phosphate from IMP1 (3-5) shows a possible role of IMP as an intermediate in the biosynthesis of other purine nucleotides.

In order to investigate the possibility of the enzymic forma- tion of guanosine nucleotides from IMP a study of the metab- olism of this substance in extracts of pigeon liver was carried out. It appeared probable that the introduction of an amino group in Position 2 of IMP would be preceded by an oxidation of this position to give a xanthine nucleotide. This hypothesis was further strengthened by the finding of Magasanik and Brooke (6) that a guanineless auxotroph of Aerobacter aerogenes accumulated xanthosine.

When C14-labeled IMP was incubated with a crude extract of pigeon liver in the presence of oxidized DPN a new C4-labeled compound appeared that could be identified as XMP. Upon incubation of this substance with the crude extract together with adenosine triphosphate, Mg++ ions, and n-glutamine or n-glutamate, a compound identified as GMP was formed. Fur- ther experiments implicated n-glutamine rather than n-gluta- mate as the amino group donor in the amination of XMP to GMP. These results are summarized in Scheme I.

IMP = XMP ~ Adenosine triphosphate

n-glutamine p GMP

SCHEME I

A preliminary report of these findings has appeared elsewhere (7). Independent of our work, Abrams and Bentley (8, 9) were able to show the same reaction in extracts of rabbit bone marrow, and Gehring and Magasanik (10) and Magasanik et al. (11) partially purified an enzyme from A. aerogenes that catalyzed the oxidation of IMP to XMP. Moyed and Maga- sanik have later reported on the purification of an enzyme from this microorganism that aminates XMP to GMP with ammonia as amino group donor instead of n-glutamine (12).

* This investigation has been sponsored by grants from the Swedish Medical Council.

1 The abbreviations used are : AMP, adenosine 5’-phosphate; ATP, adenosine 5’-triphosphate; DNA, deoxypentose nucleic acid; DPN, diphosphopyridine nucleotide; GMP, guanosine Q’-phos- phate; GTP, guanosine 5’-triphosphate; hexose-di-P, hexose di- phosphate; IMP, inosine 5’-phosphate; PNA, pentose nucleic acid; TPN, triphosphopyridine nucleotide; Tris, tris(hydroxy- methyl)aminomethane; UMP, uridine 5’-phosphate; and XMP, xanthosine 5’-phosphate.

MATERIALS AND METHODS

Reagents-4-Amino-5-imidazole carboxamide-HCl, AMP, ATP, DPN (90 per cent pure), and GMP were products of the Sigma Chemical Company, hexose-di-P, 3-phosphoglyceric acid and ribose 5-phosphate (barium salt) were obtained from Schwarz Laboratories, Inc., and pyridoxalphosphate (70 per cent pure) from Versatile Chemicals, Inc. Urea-Cl4 was purchased from the Radiochemical Centre, Amersham, England.

IMP was prepared from AMP by enzymatic deamination with AMP deaminase prepared from rabbit muscle according to Kalckar (13). IMP was crystallized as Ba-salt after chro- matography on Dowex 2 with 2 N formic acid.

IMP-2 ,8-Cl4 was obtained by incubating homogenates of pigeon liver with formate-Cl4 as described by Greenberg (14). It was purified by chromatography on Dowex 2 with formic acid and crystallized as Ba-salt to constant radioactivity after addition of carrier IMP.

XMP was obtained from GMP by deamination with nitrite (15). It was precipitated from the reaction mixture as Ba-salt, dissolved in dilute HCl and Ba++ ions removed with Na2S04. The crude XMP was purified by chromatography on Dowex 2 with 2 N formic acid and finally precipitated as the Ba-salt.

The mixed 2’- and 3’-isomers of XMP were obtained by deamination of the mixed 2’- and 3’-isomers of GMP (obtained from Dr. P. Reichard) as described above for the preparation of XMP.

XMP-Cl4 was in the first experiments obtained by the en- zymatic oxidation of IMP-C14 but was later synthesized en- zymatically from xanthine-2-C14 in the following way. An enzyme extract was prepared from 3 gm. of lyophilized Esch- erichia coli by homogenization in 50 ml. of 0.05 M phosphate buffer, pH 7.4, in a Potter homogenizer and vibration with 0.25 mm. ballotini in a microid shaker for 2 hours. After re- moval of the ballotini by filtration the preparation was centri- fuged at 105,000 x g for 45 minutes. All operations were carried out at +2”. The supernatant solution was incubated at 37” for 30 minutes with 150 pmoles of xanthine-2-Cr4, 600 pmoles of ribose 5-phosphate, 50 pmoles of ATP, 3000 pmoles of MgS04, and 3000 pmoles of 3-phosphoglyceric acid. After deproteinization with HC104, neutralization with KOH, and removal of KCIOl by centrifugation the XMP-2-Cl4 was ob- tained by chromatography on Dowex 2 with formic acid. The yields of XMP calculated on xanthine varied considerably from 25 per cent down to about 5 per cent. Attempts to syn- thesize XMP from xanthine with extracts of pigeon liver or rat liver were unsuccessful. No attempts were made to study the ribotidation of xanthine in more detail.

138

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July 1958 U. Lagerkvist 139

Xanthine-2-Cl4 was synthesized by condensation of 4-amino- 5-imidazole carboxamide-HCl with urea-U4 (16). 38 mg. of 4-aminod-imidazole carboxamide-HCl were carefully mixed with 10 mg. of urea and heated at 175” in an oil bath for 2 hours. The hard lumps obtained were extracted with about 10 ml. of water as the pH was adjusted to 8 to 9 with alkali. After add- ing enough concentrated HCl to the extract to make it 0.5 N

with respect to HCl, xanthine-Cl4 was purified by chromatog- raphy on Dowex 50 with 1 N HCI (17) and crystallized. The yield of xanthine calculated on urea was 60 per cent.

Chromatographic Separation of Puke Nucleotides-Chroma- tography on Dowex 2 in formate form by gradient elution with formic acid and ammonium formate buffer according to Hurl- bert et al. (18) was used.

Pentose was determined by the method of Mejbaum (19). Inorganic phosphate was determined according to Lowry

and Lopez (20). Determinations of Cl4 were made in a Tracerlab windowless

gasflow counter. The samples were plated on aluminum disks. Extracts of Pigeon Liver-Pigeon livers were removed and

immediately cooled in ice. After being minced coarsely they were homogenized with 10 volumes of cold acetone (-10”) in a Waring Blendor, filtered, and the filter cake resuspended in cold acetone and homogenized again. The acetone powder was dried in vacua after filtration. Acetone powder prepared in this way could be stored in the cold room at least 2 or 3 months without loss of activity. Extracts were prepared by homogenizing the acetone powder with 10 to 15 volumes of 0.05 M phosphate buffer, pH 7.4, in a Potter homogenizer. After stirring for about 1 hour the extract was centrifuged at 20,000 x g for 15 minutes and the slightly turbid supernatant solu- tion dialyzed overnight against 0.01 M phosphate buffer, pH 7.4. All operations were carried out at $2”.

RESULTS

Preliminary Experiments-When dialyzed extracts of pigeon liver prepared as described above were incubated with IMP- 2,8-C**, ATP, hexose-di-P, DPN, nicotinamide, pyridoxalphos- phate, n-aspartate, n-glutamate, n-glutamine, NH4C1, and Mg++ ions as described in Experiment 14 of Table I, two new radio- active compounds were obtained after chromatography of the deproteinized reaction mixture on Dowex 2 with formic acid. The first radioactive peak appeared in the position expected for GMP whereas the second was located between IMP and ADP in a position later shown to be occupied by XMP. The new compounds were found to be inseparable from authentic GMP and XMP respectively by chromatography on Dowex 2 with formic acid or ammonium formate buffer, pH 5.0. On acid hydrolyses they gave rise to compounds that behaved as gua- nine and xanthine respectively when subjected to chromatog- raphy on Dowex 50 with HCl. After adding a large excess of nonradioactive guanine or xanthine the radioactive hydrolysis products could be crystallized to constant radioactivity. On incubation with crude crotalus venom 5’-phosphatase the two compounds were completely dephosphorylated. They were therefore tentatively identified as GMP and XMP respectively. For a full identification of the compounds see below.

Identijication of the New Compounds as GMP and XMP- For the identification of the compound that was thought to be identical with GMP an extract was prepared from acetone pow- der of pigeon liver by extraction with 0.05 M Tris-buffer, pH

7.4. The extract was precipitated by saturation to 80 per cent with ammonium sulfate. The precipitate was dissolved in 0.05 M Tris-buffer, pH 7.4, and dialyzed overnight against 100 volumes of 0.01 M Tris-buffer of the same pH. This enzyme preparation was incubated with XMP, ATP, n-glutamine and Mg++ ions in a T&,-buffer, pH 7.4, and the resulting compound isolated by chromatography on Dowex 2 with formic acid. The compound was found to have a light absorption maximum at 256 rnp in 0.01 N HCI and on analyses guanine, ribose, and phosphate appeared in the molar ratio 1.08 : 1.03 : 1.00. On incubation with crude crotalus venom 5’-phosphatase it was completely dephosphorylated under conditions when only negligible dephosphorylation of the mixed 2’- and 3’-isomers of guanylic acid occurred.

To characterize the compound tentatively identified as XMP an enzyme extract prepared as described above was incubated with IMP, oxidized DPN and nicotinamide in a Tris buffer, pH 7.4. The resulting compound was purified by chromatog- raphy on Dowex 2 with formic acid, and after another chro- matography on Dowex 2 with formic acid it was found to have an absorption maximum at 263 rnp in 0.01 N HCl and to contain xanthine, ribose, and phosphate in the molar ratio 1.05 :0.97 : 1.00. On incubation with crude crotalus venom 5’-phosphatase the compound was completely dephosphorylated although the mixed 2’- and 3’-isomers of xanthidylic acid were practically unaffected.

Formation of XMP from IMP-In order to investigate more closely the formation of XMP from IMP the experiments re- ported in Table I were carried out.

The oxidation of IMP to XMP required oxidized DPN, and

TABLE I The influence of ATP and DPN on formation

of XMP and GMP from IMP

In Experiments 14 to 16 a dialyzed extract of 566 mg. of pigeon liver acetone powder was incubated at 37” for 60 minutes with 1 pmole of IMP-2,8-C14 (450,006 c.p.m. per pmole), 4 pmoles of ATP, 100 pmoles of hexose-di-P, 20 pmoles of DPN, 120 pmoles of nicotinamide, 150 pg. of pyridoxal phosphate, 120 pmoles of n-as- partate, 240 pmoles of n-glutamate, 60 pmoles of L-glutamine, 120 pmoles of ammonium chloride, 120 pmoles of MgS04 and 1000 pmoles of phosphate buffer, pH 7.4, with the modifications indi- cated in the table. The final volume was 20 ml.

In Experiment 20 a dialyzed extract of 506 mg. of acetone pow- der was incubated at 37” for 60 minutes with 1 pmole of IMP-2,8- Cl* (450,000 c.p.m. per Imole), 20 pmoles of DPN, 120 pmoles of nicotinamide and 1000 pmoles of phosphate buffer, pH 7.4, in a final volume of 15 ml.

The reactions were stopped by heating for 3 minutes in a boil- ing water bath and the precipitating protein centrifuged off. The purine nucleotides were separated by chromatography on Dowex 2 according to Hurlbert et al. (18). IMP, XMP, and GMP were determined by their radioactivity.

___- Modifications of medium

Experiment No. XMP formed GMP formed DPN ATP

Nicotinamide Hexose-di-P

~moles pmoles

14 + + 0.19 0.18 15 - + 0.00 0.00 16 + - 0.33 0.01 20 0.44 0.01

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140 Xunthosine 5’-Phosphate as Intermediate Vol. 233, No. 1

TABLE II The influence of ATP, ammonia and some amino acids on ami-

nation of XMP to GMP

In Experiments 23 to 24 a dialyzed extract of 500 mg. of pigeon liver acetone powder was incubated at 37” for 60 minutes with 0.36 pmoles of XMP-2-W (220,000 c.p.m. per pmole), 2 pmoles of ATP, 100 pmoles of 3-phosphoglyceric acid, 200 pg. of pyridoxal phosphate, 120 pmoles of n-aspartate, 240 pmoles of n-glutamate, 60 rmoles of n-glutamine, 120 pmoles of ammonium chloride, 120 pmoles of MgSOb and 1000 rmoles of phosphate buffer, pH 7.4, with the modifications indicated in the table. The final volume was 20 ml.

In Experiments 29 to 32 a dialyzed extract of 500 mg. of ace- tone powder was incubated at 37” for 60 minutes with 0.20 pmoles of XMP-2-W (220,000 c.p.m. per pmole), 2 pmoles of ATP, 100 rmoles of 3-phosphoglyceric acid, 200 pg. of pyridoxalphosphate, 120 pmoles of MgSOa, 1000 pmoles of phosphate buffer, pH 7.4, and 100 pmoles of either n-aspartate, n-glutamate, n-glutamine or ammonium chloride in a final volume of 15 ml. The reaction was stopped and the purine nucleotides prepared as described in Table I.

Exper;yent

23 24 29 30 31 32

ATP 3- Phospho-

glayy

Modifications of medium

L-Aspar- L-Gluta- tate mate

+ + + - - -

+

+ -

+ -

-

L-GlUta- mine

+ + - -

+ -

N&Cl

- I

GMP formed

jm&oles

0.17 0.00 0.01 0.09 0.14 0.02

ATP was not necessary for the reaction. Nicotinamide was included in the medium in order to inhibit the breakdown of DPN.

In Experiment 14 of TabIe I, GMP was formed together with XMP but in Experiment 15 when DPN was excluded from the medium there was no formation of GMP. This would indicate that XMP is an obligatory intermediate in the forma- tion of GMP from IMP in extracts of pigeon liver.

An attempt was made to decide whether DPN or TPN was involved in the oxidation of IMP. It was found that at low concentrations DPN was at least 10 times as effective as TPN in promoting the formation of XMP from IMP (Pig. 1).

Formation of GMP from XMP-The experiments reported in Table II were carried out in an attempt to investigate the role of ATP in the amination of XMP and to differentiate between n-aspartate, n-glutamate, n-glutamine, and ammonia as amino group donors.

The amination reaction required ATP and Mg++ ions while pyridoxalphosphate that was included in the medium was later shown to be without effect in the reaction. 3-Phosphoglyceric acid was included in order to keep the ATP-level as constant as possible.

From Experiments 29 to 32 it can be seen that n-glutamine and n-glutamate under these conditions gave approximately the same synthesis of GMP, whereas n-aspartate and NH2 ions were inactive. In order to distinguish between glutamine and glutamate as amino group donors, their ability to promote GMP

I__I I I 1 2

MOLARITY OF DPN AND TPN xl ti FIG. 1. Dependence of IMP oxidation on DPN and TPN. A

dialyzed extract of 500 mg. of pigeon liver acetone powder was incubated at 37” for 30 minutes with 0.20 Hmoles of IMPS,%04 (1.5 X lo6 c.p.m. per @mole), 50 pmoles of nicotinamide, 500 pmoles of phosphate buffer, pH 7.4, and DPN or TPN as indicated in the figure. The final volume was 5 ml. The reaction was stopped and the purine nucleotides were prepared as described in Table I. O-----C = XMP formed in the presence of DPN; O-----O = XMP formed in the presence of TPN.

synthesis was investigated at decreasing concentrations of the amino acids (Fig. 2).

These experiments showed that at low concentrations L- glutamine gave a GMP synthesis that was about 8 times that obtained with n-glutamate. This result implicated n-glutamine rather than n-glutamate as the specific amino group donor and this conclusion was later confirmed by experiments with puri- fied aminating enzyme (21). The purified preparation required n-glutamine and was completely inactive with n-glutamate. The effect of glutamate in the crude extracts must therefore be attributed to the presence of glutamine synthetase in these preparations.

DISCUSSION

Mainly through the work of Greenberg (1) and Buchanan and coworkers (2) the biosynthesis of IMP has been elucidated in great detail. Although interest in the metabolic behavior of this compound first arose with the indication of its key role in the excretory mechanism of uricotelic organisms, the possi- bility that it might also be involved in the formation of adenine and guanine nucleotides seemed worth investigating. Recently Carter and Cohen (3, 4) have shown the reversible formation of adenylosuccinate from fumaric acid and AMP with a purified enzyme fraction from yeast. Lieberman (5) has later purified an enzyme from Escherichia coli that catalyzes the condensation of n-aspartate and IMP to adenylosuccinate in the presence of GTP. A synthetic pathway from IMP to adenosine 5’-nucleo- tides has thereby been established.

The results reported above and those obtained by Abrams

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July 1958

2 4 6 8

U. Lagerhwist

MOLARITY OF L-GLUTAMINE

AND L-GLUTAMATE x IO4 FIG. 2. Dependence of the amination of XMP to GMP on

n-glutamine and n-glutamate. A dialyzed extract of 300 mg. of pigeon liver acetone powder was incubated at 37” for 60 minutes with 0.30 rmoles of XMP-2-W (220,000 c.p.m. per pmole), 2 &moles of ATP, 50 pmoles of 3-phosphoglyceric acid, 200 pg of pyridoxalphosphate, 120 pmoles of MgSOe, 1000 pmoles of phos- phate buffer, pH 7.4, and n-glutamine or n-glutamate as indicated in the figure. The final volume was 13 ml. The reaction was stopped and the purine nucleotides were prepared as described in Table I. 0-O = GMP formed in the presence of n-gluta- mine; 0-0 = GMP formed in the presence of L-glutamate.

and Bentley (8, 9) and Magasanik et ~2. (10-12) have shown the occurrence of a synthetic pathway from IMP to GMP by way of XMP both in animal tissues and microorganisms. The main difference between the enzymes of animal origin and those from microorganisms seems to be the utilization of different amino group donors in the amination of XMP. The results of investigations on stoichiometry and reaction mechanism of the amination reaction with a purified enzyme fraction from pigeon liver will be presented in a subsequent paper (21).

The oxidation of IMP to XMP required DPN. The small activity obtained with TPN may be explained by the formation of DPN from TPN. Regarding the mechanism of the reaction the most reasonable hypothesis seems to be the intermediate formation of a hydrated compound followed by the removal of hydrogen by DPN.

The discovery of biosynthetic mechanisms for the formation of AMP and GMP from IMP in animal tissues and micro- organisms poses the question of the possible role of these com- pounds in nucleic acid synthesis. The work of Hurlbert and Potter (22) with rat liver has implicated UMP rather than the 2’- or 3’-isomers as intermediate in the biosynthesis of PNA pyrimidine nucleotides. Recently Ochoa and coworkers (23, 24) have shown the formation of a PNA-like polynucleotide from pyrimidine and purine nucleoside 5’diphosphates with an enzyme fraction from Azotobacter z&elan&i, and Kornberg et al. (25, 26) reported on the polymerization of the 5’-triphos- phates of thymidine, deoxycytidine, deoxyadenosine, and deoxyguanosine to a DNA-like product with an enzyme prep- aration from E. coli in the presence of preformed DNA as a primer.

These results are consistent with the hypotheses that py- rimidine and purine nucleoside 5’-phosphates and deoxynucleoside 5’-phosphates are intermediates in the biosynthesis of the nucleic acids. The results reported above, together with the indication of the formation of AMP from IMP, may therefore represent a link between the reaction sequence leading to hy- poxanthine formation and the biosynthesis of PNA purine nucle- otides.

SUMMARY

An enzymic synthesis of guanosine 5’-phosphate from inosine 5’-phosphate has been shown in crude extracts of pigeon liver. Xanthosine 5’-phosphate is an obligatory intermediate in this reaction. The oxidation of inosine 5’-phosphate to xanthosine $-phosphate was shown to be dependent on diphosphopyridine nucleotide. The amination of xanthosine 5’-phosphate to guanosine 5’-phosphate required adenosine triphosphate and n-glutamine or n-glutamate. Evidence is presented to show that n-glutamine rather than L-glutamate is the actual amino group donor.

Acknowledgments-The skilled technical assistance of Mrs. B. Edling and Mrs. G. Degerstedt is gratefully acknowledged.

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Ulf LagerkvistAS AN INTERMEDIATE

Biosynthesis of Guanosine 5'-Phosphate: I. XANTHOSINE 5'-PHOSPHATE

1958, 233:138-142.J. Biol. Chem. 

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