BIOSYNTHESIS OF THE PURINES

XII. STRUCTURE, ENZYMATIC SYNTHESIS, AND METABOLISM OF 5-AMINOIMIDAZOLE RIBOTIDE*

BY BRUCE LEVENBERGt AND JOHN M. BUCHANAN

(From the Division of Biochemistry, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts)

(Received for publication, July 11, 1956)

The isolation and identification of two ribotide derivatives of glycinamide arising as products of enzymatic reactions of pigeon liver extract directed toward purine biosynthesis have recently been reported (2, 3). The com- pounds, glycinamide ribot,ide (GAR) and (Lu-N-formyl)-glycinamide ribo- tide (FGAR), were demonstrated to be intermediates in the synthesis of inosinic acid (IMP) de novo. With the aid of the antibiotic, r,-azaserine, which exert.ed a strong inhibitory effect on puke format.ion at a step subsequent to the synthesis of FGAR, it was possible to accumulate rela- tively large amounts of the latter compound and to study its fate in further reactions of IMP biosynthesis (4).

In this paper, evidence is presented for 6he reaction of FGAR with glutamine and adenosine kiphosphate (ATP) to yield a new arylamine ribotide. This compound has been isolated and identified by chemical analysis as 5-aminoimidazole ribotide (AIR).

Methods and Materials

Chemical Determinatiorw-The met,hods employed for the chemical analysis of AIR are identical to those described in Paper XI (4). Owing to the relative lability of AIR, it was necessary to carry out some of the analyses of this compound with freshly prepared solutions.

Assag for Synthesis of AIR-The formation of AIR was conveniently followed by means of the react,ion for arylamines as described by Bratt.on and Marshall (5), except that the volume of t,he aliquot of reaction mixture and the quant,it,ies of reagents used were one-tenth those originally recom- mended. Furt,hermore, the solutions were acidified with t,richloroacetic acid (TCA) rather than with H,SOI. The enzymatic reaction, usually

*A preliminary report of this work has been published (1). This work has been support.ed by grants-in-aid from the National Cancer Institute, National Institutes of Health, United States Public Health Service, the Damon Runyon Memorial Fund for Cancer Research, Inc., and the National Science Foundation.

t United States Public Health Service Research Fellow of the National Institute of Neurological Diseases and Blindness (1954-55).

1005

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1006 BIOSYNTHESIS OF PURINES. XII

0.5 ml. in volume, was t.erminated by the addition of 0.2 ml. of 30 per cent TCA after the incubation vessels had been chilled briefly in an ice bath. Denatured protein was removed by centrifugation. A suitable aliquot of the supernatant solution was transferred to a small test tube and diluted with water to 0.55 ml. in preparation for the arylamine deter- mination. The color which developed was read after Q hour in the Beck- man model DU spectrophotometer fitted with an attachment for the use of microcells. The measurement of the optical density was carried out at

2.l-

1.8 -

I J 400 450 500 550 . 600

WAVE LENGTH (mp)

Fro. 1. Absorption spectra of the chromophores from the diazotization and coup- ling of 5-aminoimidazole ribotide (0 ) and 5-amino-4-imidazolecarboxamide ribotide (0). The Bratton and Marshall reaction (5) was performed as described under “Methods and materials.” Absorption of light was measured with the use of the microcell attachment of the Beckman model DU spectrophotometer.

500 rnp, the wave length at which the orange chromophore produced in this reaction absorbed maximally (see Fig. 1). This derivative of 5-amino- imidazole ribotide has a molecular extinction coefficient of 24,600.

Assay for Synthesis of IMP from AIR-The method of Schulman, Sonne, and Buchanan (6) was used, with a slight modification, for the determi- nation of the extent of conversion of CY4-labeled AIR to IMP. The alter- ation in the procedure consisted of the replacement of most of the chloride content of the incubation mixture by an equivalent amount of sulfate ion. This obviated the necessity for the time-consuming recrystallization step heretofore employed.

The source and preparation of many of the materials have been de- scribed in Paper XI (4).

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B. LEVENBERG AND J. M. BUCHANAN 1007

Preparation of Cl’-LabeM Ureidoimidazole Picrate-Unlabeled ureido- imidazole was prepared in methanolic solution from 5-aminoimidazole (7) and potassium cyanate by an adaptation of the procedure of Hunter and Hlynka (8). To this solution was added the reaction product of AIR-2-C” and potassium cyanate obtained by a similar procedure. The mixture was lyophilized to dryness and the residue was extracted several times with water until most of the desired materials, as determined by the Pauly reaction, had been brought into solution. The derivative of AIR in this extract was hydrolyzed with 2 N HCl at 100’ for 1 hour and the hydrolysate was placed on a Dowex 50 potassium column (0.8 X 11 cm.). Ureido- imidazole, which remained on the column, was then eluted with 0.015 M potassium phosphate buffer, pH 6.5. The ureidoimidazole-containing fractions were located with the Pauly reagent, pooled, and lyophilized to a small volume. Potassium ions were removed by careful addition of perchloric acid in the cold. To this acid solution, free of potassium ions, were added 65 mg. of picric acid. After chilling, the picrate was precipi- tated and recrystallized from a small volume of hot water; m.p. = 200- 201’ (uncorrected); m.p. reported by Hunter and Hlynka (8) = 200”. The ureidoimidazole picrate, which was plated for determination of radio- activity, was recrystallized from hot water before each succeeding deter- mination.

EXPERIMENTAL

An indication of the possible nature of chemical events taking place in the biosynthesis de mo of IMP subsequent to the formation of FGAR was first obtained from consideration of some of the factors involved in the metabolic reactions concerned with the completion of the purine ring sys- tem from the aliphatic ribotide. The requirement for aspartic acid, glutamine, and CO* in these reactions (3) with pigeon liver enzymes was in complete agreement with previous experiments with isotopic tracers which had demonstrated that N1, Na, and Cs of the purines were derived metabolically from these substrates, respectively (9). It was found, in addition, that azaserine inhibited markedly the conversion of FGAR to IMP. Since GAR and FGAR were the only two intermediates shown to accumulate when pigeon liver enzymes were inhibited with azaserine (4), it was assumed that azaserine affected the synthesis of inosinic acid by blocking the enzymatic reaction concerned with the initial step of the further metabolism of FGAR.

If the inhibitory effect of azaserine could be overcome by elevation of the concentration of any one of the above three substrates, it was felt that this substance would participate in the initial reaction of FGAR. The results of such an experiment are shown in Table I.

It is seen that, of the three substrates which were necessary for the re-

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1008 BIOSYNTHESIS OF PURINES. XII

action, the addition of glutamine at increased levels resulted in marked reduction of the inhibitory effect of azaserine. This finding provided evidence of an indirect nature that the introduction of what would corre- spond to nitrogen atom 3 of the purine ring contituted the first “group transfer” reaction in the further metabolism of FGAR.

Direct experimental verification of this hypothesis was obtained by incubation of substrate quantities of FGAR with glutamine and ATP in the presence of an enzyme system of pigeon liver extract. The product of these reactions was a new arylamine ribotide which, upon reincubation with aspartic acid and a small amount of bicarbonate, was converted to

TABLE I Ability of L-Cl&amine to Overcome Inhibitory Action of L-Azaserine

in Synthesis 0f IMP from FGAR V

Vessel No. Aaserble GlUtamine Aspartic acid Potassium bicarbonate

p?nolss PmqlOS pmolw

1 15 15 2 2.8 15 15 3 2.8 15 75 4 2.8 75 15 5 2.8 15 15 6 8.4 75 15

~molos

15 15 15 15 75 15

-

IMP synthesized

wnole

0.30 0.09 0.08 0.21 0.09 0.09

Each vessel contained, in a final volume of 1.00 ml., the following amounts of ma- teriais: 0.4 pmole of Ba FGAR-l-04; 30 pmoles of sodium formate; 15 pmoles of so- dium 3-phosphoglyceric acid; 20 &moles of KgSO4; 3 pmoles of MgSO4; 12 rmoles of sodium phosphate buffer, pH 7.4; “0 to 45” ethanol-precipitated enzyme, equivaicnt to 1.3 ml. of pigeon liver extract. The vessels were incubated for 1.5 hours at 38”. IMP was determined according to the procedure described under “Methods and materials.”

IMP via the formation of 5-amino+Limidazolecarboxamide ribotide. In the following section are described methods for the preparation of

AIR from both glycine (i.e. synthesis de novo) and FGAR. For reasons not fully understood at present, t.he product prepared by incubation of pigeon liver enzymes wit,h glycine was unstable when attempts were made to isolate it. However, when FGAR was used as substrate for the syn- thesis, the product, though unstable, could be isolated. With this latter procedure it has been possible to purify and characterize by chemical analysis this new arylamine compound.

Enzymatic Synthesis and Isolation of AIR

Preparation of AIR de Noon-It was desirable for later enzymatic studies to develop a method of synthesis of AIR on a relatively Iarge scale which

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B. LEVENBERG AND J. M. BUCHANAN 1009

would obviate the need for the time-consuming preparation and isolation of FGAR and make use of substrates which were more readily available. Accordingly, the possibility of effecting the synthesis de lzovo of AIR was investigated. Owing to the presence of interfering enzymatic components in the “0 to 13” ethanol fraction of pigeon liver extract and to traces of substrates in this fraction which participate in the further metabolism of AIR, the synthesis of AIR de nova proved successful only after this lower enzyme fraction was omitted from the system employed. Under these circumstances, it became necessary to add substrate quantities of li-phos- phoribosyl pyrophosphate (PRPP), the compound synthesized by a kinase present in the “0 to 15” ethanol fraction (10) and required for the initial ribotidation reaction in purine synthesis de novo (3). Although excellent formation of AIR was found to occur upon incubation of the enzymatic and chemical components described below, repeated attempts to isolate the product were unsuccessful. The following method has been of con- siderable use, however, in cases in which it was desired to prepare large quantities of AIR in situ for use in the study of its further metabolic re- actions.

Incubation Procedure-Each vessel contained, in a final volume of 60 ml., the following quantities of materials expressed in micromoles: glycine 375, L-glutamine 375, sodium PRPP 58, disodium ATP 29, sodium formate 187, sodium 3-phosphoglycerate 560, sodium phosphate buffer, pH 7.4, 1480, potassium chloride 4400, magnesium chloride 1850, and 310 mg. of lyophilized “13 to 33” ethanol fraction of pigeon liver extract (for prepara- tion of the enzyme system see the following section).

Incubation was carried out for 1 hour at 38”. The solution, obtained after removal of protein by heat denaturation at lOO”, contained approxi- mately 25 pmoles of AIR per vessel.

Preparation of AIR from FGAR

Enzyme System-Pigeon liver extract was prepared from livers of freshly killed pigeons in the manner described in Paper I (6), with the exception that bicarbonate was omitted from the mixture of internal salts. The protein which precipitated at -6” from this extract upon addition of cold ethanol to a concentration of 13 per cent was removed by centrifugation. The supernatant solution was again treated with 90 per cent ethanol at -18’ and the protein which precipitated between a concentration of 13 and 33 per cent ethanol was removed by centrifugation, dissolved in a small volume of water, and lyophilized. The resulting “13 to 33” ethanol fraction remained active for several months when stored at -15”.

Incubation Procedure-Each vessel contained, in a final volume of 17 ml., the following quantities of materials, expressed in micromoles: the barium salt of FGAR-1-W 5, L-glutamine 190, disodium ATP 24, sodium

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1010 BIOSYNTHESIS OF PURINES. XII

phosphate buffer, pH 7.4, 600, potassium sulfate 650, magnesium sulfate 100, magnesium chloride 80, and 87 mg. of lyophilized “13 to 33” ethanol fraction of pigeon liver extract.

Ten such vessels were incubated for 1 hour at 38” and the reaction mixtures were then pooled in one large flask. Protein was coagulated by heating the contents of the flask at 100” for 3.5 minutes. The suspension was chilled in an ice bath and the precipitated protein was removed by centrifugation at 3”. Based upon the FGAR added to the enzymatie system, the yield of AIR, as determined by performing the Bratton-Mar- shall reaction on an aliquot of the supernatant solution, was approximately 80 per cent.

Isolation of AIR by Chromatography and Preparation of Crude Barium Salt

The supernatant solution from the deproteinized incubation mixture was adjusted to pH 8 with a few drops of 4 N KOH and then placed, in equal volumes, upon eleven columns of Dowex 1 acetate (0.8 X 11.4 cm.) at 3”. The resins were then washed with water and eluted rapidly with 0.04 M

ammonium acetat,e buffer, pH 5.3. Fractions containing approximately 20 ml. were collected manually. Suitable aliquots were then removed from each fraction and assayed for the presence of the arylamine com- pound. The pattern of elution of AIR from Dowex 1 acet.ate under these conditions is shown in Fig. 2. Fractions from all columns containing the amine were pooled and the resulting solution was rapidly concentrated in vacua to 2 ml. in a “Rinco” laboratory evaporator.1

The barium salt of AIR was precipitated in impure form by the addition of 300 pmoles of barium acetate and 38 ml. of absolute ethanol to the aryl- amine concentrate. The suspension was placed at -15” overnight and the precipitate was collected by centrifugation. It was washed once with 90 per cent ethanol, once with ether, and dried in vacua.

PurQication of Barium Salt-80 mg. of the crude barium salt were dis- soIved in 28 ml. of 0.02 N HCI and this solution was placed upon a column of Dowex 50 sodium (2.5 X 3.8 cm.) at 3”. The major portion of the AIR applied to the resin passed through the column and was collected in the first fraction. The resin was washed briefly with 10 ml. of water and this second fraction was combined with that obtained initially. This proce- dure succeeded in removing glutamine (and perhaps glutamic acid), which was the chief impurity present in the initial product isolated as the barium salt.

The pH of the solution was adjusted to 8 with a few drops of 4 N KOH

1 Manufactured by the Rinco Instrument Company, Greenville, Illinois, and dis- tributed by the Aloe Scientific Division of the A. S. Aloe Company, 5655 Kingsbury Street, St. Louis 12, Missouri.

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B. LEVENBERG AND J. hf. BUCHANAN 1011

and the AIR in this fract,ion was again isolated from a column of Dowex 1 acetate (0.8 X 14 cm.) at 3” in a manner similar to that described above. The fractions of the eluate which contained arylamine were pooled and evaporated to 3 ml. as before. The barium salt which was obtained upon the addiCon of 50 rmoles of barium acetate and 8 volumes of absolute eth- anol was permitted to precipitate overnight at -15”. It was collected by centrifugation, washed repeatedly with cold 95 per cent ethanol and finally with ether, and dried in vucuo over PzOs. The over-all yield of the

I J l-L-4 . . , I

‘r’ I 2 3 4 5 6 7 8 9 I;,

BUFFER FRACTION NUMBER

FIG. 2. Elution of AIR from a column of Dowex 1 acetate. The chromatographic methods used are described in the text. The buffer used for elut.ion was 0.04 M am- monium acetate, pH 5.3. Elution of the arylamine ribotide was followed by reaction of an aliquot. (usually 0.40 ml.) from each fraction with the reagents of Bratton and Marshall (5) as described under “Methods and mat.erials.” Spectrophotometric measurement at 500 * was made of the chromophore produced by reaction of the Bratton and Marshall reagents wit.h AIR.

purified barium salt of AIR was 25 per cent (based upon the quantity of FGAR present in the incubation mixture). This material, which was pale green in color, was used for the chemical analyses reported below.

It can be calculat,ed on the basis of the molecular weight of the barium salt of AIR (assuming no water of hydration) that the two samples sub- jected to analysis were approximately 35 and 60 per cent pure, respectively. The losses encountered because of the considerable instability of this arylamine ribotide have hampered further attempts at removal of the re- maining impurities, which are believed to consist chiefly of inorganic salts.

Analysis and Chemical Properties of AIR-Glycine, formic acid, and ammonia were found by Hunter and Nelson (7) to be liberated as products

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1012 BIOSYNTHESIS OF PURINES. XII

of hydrolysis of the aminoimidaaole structure in strongly acid or alkaline solutions. In their studies these substances appeared in the approximate molecular ratios of 1: 1:2, respectively. By making use of this informa- tion, it has been possible to extend analytical studies upon AIR to include determinations of these products as well as of pentose, organic phosphate, and total nitrogen.

The results of a chemical analysis of the components liberated upon hydrolysis of two different samples of the barium salt of AIR are shown in Table II. In the second and third columns these data are expressed on the basis of the number of micromoles of substances determined per mg. of Samples a and b, respectively. When these values are compared

TABLE II Chemical Analysis of b-Aminoimidazole Ribotide

Ba salt of AIR

Analysis* Sample a Sample b Sample B Sample b

pwwles per mg. pmoles per m*.

Glycine....................... 1.41 1.00 Pentose...................... 0.525 1.41 1.00 1.00 Organic P.................... 0.552 1.80 1.05 1.26 Inorganic P . . . . . 0.024 0.00 TotalP...................... 0.576 1.30 Formate...................... 0.641 1.57 1.22 Acid-labile N.. . 1.14 3.21 2.18 Total N...................... 4.63

1.10 2.27 3.23

* The methods of analysis are given under “Experimental.”

to those of glycine taken as unity (or pentose, as in the case of Sample a), the results assume the characteristics of molecular ratios of the components to that of the glycine (or pentose) residue. These ratios are presented in the fourth and fifth columns of Table II.

It is seen from the results reported here that glycine, formate, pentose, organic phosphate, acid-labile nitrogen, and total nitrogen were formed in the approximate molecular ratios of 1: 1: 1: 1: 2: 3 as products of hydrolysis or combustion of AIR.2

* Since the sample was purified by chromatography on a Dowex 1 acetate column and by precipitation by addition of barium acetate, it was possible that barium 5-aminoimidazole ribotide could precipitate as the acetate salt. In order to test this, a sample was kindly assayed by Dr. J. C. Rabinowitz for its content of acetate by the enzymatic method of Rose et al. (11). It was found that the sample contained 0.5 pmole of acetate per pmole of aminoimidazole ribotide. It was, therefore, diffi- cult to determine whether the acetate was present as an impurity or as a salt of the imidazole compound.

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B. LEVENBERG AND J. M. BUCHANAN 1013

Further chemical evidence in support of the structure of AIR was ob- tained by the preparation of the ureido derivative of a sample of the ribo- tide which had been synt.hesized enzymatically from formyl-labeled FGAR and isolated by chromatography on Dowex 1 acet,ate. After admixture with synthetic, unlabeled ureidoimidazole as carrier, the ribotide was hydrolyzed and ureidoimidazole was isolated as the picrate by a proce- dure presented under “Methods and materials.” The specific activity of the picrate after each succeeding recrystallizat.ion remained essentially constant, as shown in Table III.

Solut.ions of AIR, when freshly prepared, reacted witch diazotized sul- fanilic acid in the Koessler and Hanke modificat,ion of t,he Pauly reaction (12) t.o produce an orange color which faded almost immediately. This was taken as an indication of a positive response, although the behavior of AIR in this test differed markedly from that shown by much lower

TABLE III Recrystallization of UJreidoimidazole Picrate-I-CY to Constant Speci$c Activity

No. of recrystalliitions Specific activity

c.p.m. )W $mols

1 984 2 908 3 939

The samples were corrected to infinite thinness with use of the self-absorption coefficient for uric acid.

concentrations of imidazole compounds such as histidine, which yield colors of a greater degree of stability.

Hydrolysis of AIR in the presence of acid (0.2 N HCl at 160” for 6 hours in a sealed tube) resulted in the liberation of glycine as the only ninhydrin- reactive spot detect,able on paper chromatograms (solvent system com- posed of 95 per cent ethanol-concentrated h’H40H, 95:5).

The ultraviolet absorption spectrum of AIR at pH 7 is presented in Fig. 3. No selective band of absorption of light between wave lengths 215 and 300 rnCc was observed, although a moderately &rong, general ab- sorption was evident in t,he region of lower wave lengt,hs (i.e. below 250 4.

Metabolism of AIR

When 5-aminoimidazole ribotide, which had been prepared by enzymatic synthesis from FGAR-l-CY4, was tested for its ability to function as an intermediate in t,he biosynthesis of IMP wit.h enzymes of the “13 to 33” ethanol fraction of pigeon liver extract, it was found that the radioactivity

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1014 BIOSYNTHESIS OF PUBINES. XII

of the arylamine could be converted in excellent yield to IMP. Both aspartic acid and bicarbonate were essential substrates for this reaction, and the conversion of the isotope was unaffected by the presence of a large bank of unlabeled glycine.

Further investigation of the involvement of bicarbonate in the partial synthesis of IMP from AIR revealed that CY4-labeled sodium bicarbonate was fixed in the completed purine ring during the reaction in approxi- mately the expected proportion (Table IV). Under identical experimental conditions, no evidence could be obtained for COZ fixation during the en- zymatic conversion of 5-amino4-imidazolecarboxamide ribotide to IMP.

I=

2 ‘- F

:: 9 8 I I 1

210 230 250 270 290 310

WAVE LENGTH (ffIJJ)

FIG. 3. Ultraviolet absorption spectrum of 5-aminoimidarole ribotide in neutral solution. 0.32 pmole of the barium salt of AIR (Sample b) was dissolved in 3.00 ml. of water. The absorption of ultraviolet light by this solution was measured against a blank containing 1 X 10-’ M BaCls.

Since aspartic acid, bicarbonate, and, presumably, formate were neces- sary for the completion of the purine ring from the aminoimidazole struc- ture, it was of considerable interest to determine whether further metabo- lites of AIR could be detected. It was subsequently found possible to accumulate another arylamine compound upon enzymatic reaction of AIR with aspartic acid and a small amount of bicarbonate in the presence of the “13 to 33” fraction. This new amine was easily detected by the shift in the absorption maximum of the dye produced upon reaction of depro- teinized incubation mixtures of the substrates with the reagents of Bratton and Marshall (5). In the presence of these reagents, AIR is converted into an orange chromophore (X,,, = 500 mp), whereas the second aryl- amine compound yields a purple dye (X,., = 540 mp). The absorption

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B. LEVENBERG AND J. M. BUCHANAN 1015

spectra of both chromophores are shown in Fig. 1. With the purified barium salt of AIR as a substrate, a requirement for ATP in addition to aspartic acid and bicarbonate was demonstrated. Under optimal condi- tions of enzymatic reaction, all of the AIR could be converted into the second arylamine compound.

The latter substance was found to possess far greater chemical stability than AIR and was isolated with little difficulty by passage of the depro- teinized solution from a relatively large scale incubation mixture through a Dowex 50 ammonium column, followed by chromatography of the initial eluate from this resin on Dowex 1 acetate. In contrast to AIR, this stable

TABLE IV Fixation of Cl’02 during Conversion of Atylamine Ribotide to IMP

“ifir’

1 2 3

4

P-labeled aryhmiie rihtide’ cw:t

6-Aminoimidazole ribotide ‘I “

5-Amino-4-imidazolecarbox- amide ribotide

“ ‘I

c.p.m. rmolcs

2140 + 5140 1.5 - 2460

+ 2280 0

Total &&li;tivity C”O¶ fixed er mole of IMP onned P

All vessels contained, in a final volume of 2.1 ml., the following quantities of ma- terials: 16 amoles of sodium formate, 15 pmoles of sodium 3-phosphoglyceric acid, 8 pmoles of L-azaserine, 17 cmoles of KtSO+ 4 rmoles of precipitated enzyme equiva- lent to 1 ml. of pigeon liver extract. Approximately 0.03 pmole of each ribotide was added to appropriate vessels. Vessels 1 and 2 contained 12 &moles of sodium ~-as-

partate. 37 pmoles of NaHWOs were added to Vessels 2 and 4, and a corresponding amount of unlabeled NaHCOs to Vessels 1 and 3. Incubation waz carried out at 38” for 13 hours.

* Specific activity equals 25,000 c.p.m. per pmole. t Specific activity equals 22,000 c.p.m. per rmole.

a&amine possessed an absorption band in the ultraviolet spectrum with a maximum at 267 rnp. It was shown to incorporate Nls from n-aspartic acid-N16 during its synthesis from AIR and to be converted to IMP upon incubation with serine and the “13 to 33” ethanol fraction of pigeon liver extract.

The above mentioned properties of this metabolic product of AIR were similar to those described for 5-amino-4~imidazolecarboxamide ribotide (13). Cochromatography on Dowex 1 chloride of a mixture of an authentic sample of the latter compounds with the compound formed biosyntheti- tally from AIR-5-V resulted in the elution of a single arylamine com-

* Kindly supplied by Mr. Joel G. Flaks. The sample was prepared by enzymatic reaction of 4-amino-5-imidazolecarboxamide and PRPP.

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1016 BIOSYNTHESIS OF PTJFUNES. XII

ponent. The patterns of elution of this material as measured independ- entlyby radioactivity and ultraviolet light absorption were superimposible. On the basis of the findings, the metabolic product of the reactions be- tween AIR, ATP, aspartic acid, and CO2 was ident.ified as 5-amino-4- imidazolecarboxamide ribotide.

DISCUSSION

The structural formula of AIR presented has been determined on the basis of the following t,hree considerations: (1) the results of analyses of chemical components, (2) chemical and spectral properties, and (3) bio- chemical function of t.he compound as an intermediate in t.he synt.hesis of IMP de novo.

“i Y

7\ TH NHII

‘;I CH-CHOH-CHOH-CH-CHtOPOaH2 IOI

5-Aminoimidaeole ribotide

The agreement of the molecular ratios shown in Table II with theory and the demonstrat.ion of t,he abilit,y of the arylamine to form Ei-ureido- imidazole (as its picrate derivative), provided the essential requirements for designating the structural formula of the isolated compound as 5-amino- imidazole ribotide. No information was gained, however, from these or other chemical data regarding the possibility of the existence of a carboxyl substituent at posit.ion 4 of the imidazole ring which would be removed as CO2 under the drastic condit,ions of most. of these analyses. Biochemical results did, however, furnish evidence on this point by showing that (1) bicarbonate was not required for the enzymatic synthesis of AIR from FGAR and (2) approximately 1 mole of CO2 was fixed by AIR per mole of IMP formed during the conversion of the arylamine t,o the purine com- pound. These results made it improbable that the compound isolat,ed from t,hese reactions was 5-amino-4-carboxyimidazole ribotide rather t,han 5-aminoimidazole ribotide.

The aglycone portion of AIR is apparently identical with the corre- sponding structure of the compound isolated from culture filtrates of a purine-requiring mutant of Escherichiu coli by Love and Gets (14) and identified by Love and Levenbergd as Saminoimidazole riboside. More- over, Love and Gots believe that their compound is closely related to t,he

4 Unpublished data.

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B. LEVENBERG AND J. M. BUCHANAN 1017

arylamine found by Chamberlain, Cutts, and Rainbow (15, 16) as a prod- uct of metabolism of yeast 47 grown with suboptimal levels of biotin. It is of interest that the heterocyclic ring structure of AIR has now been shown to represent an aglycone common to the pathways of purine syn- thesis de novo in avian, plant, and bacterial systems. In all probability the intracellular intermediate in all three organisms is the ribotide which is dephosphorylated by cellular systems before liberation into the medium as the riboside. The report by Rabinowitz and Pricer (17) has demon- strated that 5-aminoimidazole as the free base may likewise be involved in the catabolism of purine compounds in microorganisms.

Although the involvement of the ribotide of 5-amino+t-imidazolecarbox- amide as an intermediate in the synthesis of inosinic acid de nova has been indicated by several lines of evidence, the present report constitutes the first demonstration of the synthesis of this compound in animal tissues from known precursors of IMP. The major outlines of the biochemical mechanisms whereby the elementary carbon and nitrogen-containing pre- cursors are assembled into the final structure of a purine ribotide are now fairly evident. The detailed study of the chemistry of each of the individ- ual steps in this biosynthetic process will be of considerable interest because of the range and variability of the types of reactions involved. More detailed information on one of these steps, the conversion of FGAR to AIR, is presented in Paper XIII.

SUMMARY

A new arylamine ribotide has been isolated in the form of its barium salt as the product of a reaction between ATP, glutamine, and (&V-for- myl)-glycinamide ribotide in the presence of a soluble enzyme system of pigeon liver. The substance was identified as 5-aminoimidazole ribotide and has been shown to be an intermediate in the synthesis of inosinic acid de rwvo.

BIBLIOGRAPHY

1. Levenberg, B., and Buchanan, J. M., J. Am. Chem. floe., 78, 504 (1956). 2. Goldthwait, D. A., Peabody, R. A., and Greenberg, G. R., J. Am. Chem. Sot.,

78, 5258 (1954). 3. Hartman, S. C., Levenberg, B., and Buchanan, J. M., J. Am. Chem. SOL, 77,

501 (1955). 4. Hartman, S. C., Levenberg, B., and Buchanan, J. M., J. Biol. Chem., 231, 1957

(1956) . 5. Bratton, A. C., and Marshall, E. K., Jr., J. Biol. Chem., 128, 537 (1939). 6. Schulman, M. P., Sonne, J. C., and Buchanan, J. M., J. Biol. Chem., 198, 499

(1962). 7. Hunter, G., and Nelson, J. A., Canad. J. Res., Sect. B, 19, 296 (1941). 8. Hunter, G., and Hlynka, I., Biochem. J., 81, 488 (1937).

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Bruce Levenberg and John M. Buchanan5-AMINOIMIDAZOLE RIBOTIDE

SYNTHESIS, AND METABOLISM OFSTRUCTURE, ENZYMATIC

BIOSYNTHESIS OF THE PURINES: XII.

1957, 224:1005-1018.J. Biol. Chem. 

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