BIOSYNTHESIS OF THE PURINES

Size: px

Start display at page:

Download "BIOSYNTHESIS OF THE PURINES"

Linette Parks
5 years ago
Views:

1 BOSYNTHESS OF THE PURNES X. STRUCTURE, ENZYMATC SYNTHESS, AND METABOLSM OF (arnformyl)glycnamdne RBOTDE* BY BRUCE LEVENBERGt AND JOHN M. BUCHANAN (From the Division of Biochemistry, Department of Biology, Massachusetts nstitute of Technology, Cambridge, Massachusetts) (Received for publication, July 11, 1956) n Paper X (2), the enzyme system of pigeon liver extract responsible for the conversion of formylglycinamide ribotide (FGAR) to 5aminoimidazole ribotide (AR) was described. During the course of further study of this reaction, it was noted that the enzymatic components of the system could readily be separated into two fractions. ncubation of one of these fractions with FGAR, glutamine, and adenosine triphosphate (ATP) resulted in the formation of a new compound which, upon reaction with ATP and the second enzymatic component, was oonverted to AR. Experiments presented in this paper are concerned with the preparation and isolation in pure form of this intermediate, and with its identification by chemical methods as (aivformyl)glycinamidine ribotide (FGAM). Methods and Materiak Reference should be made to Paper X (2) for many of the chemicals, assay procedures, and analytical determinations employed here. Assay for Synthesis of FGAMThe calorimetric determination of FGAM was based upon its conversion to AR in the presence of ATP and an excess of Fraction (see under Experimental ). The arylamine was then measured by the modification of the procedure of Bratton and Marshall (3) as described in Paper X (2). Preparation of Enzymes3.5 gm. of the lyophilized 13 to 33 per cent ethanol fraction, which was capable of converting FGAR to AR, were dissolved in 230 ml. of 0.01 M sodium phosphate buffer, ph 7.4, at 3. The proteins were fractionated by the addition of solid ammonium sulfate at that temperature with stirring to insure rapid solution of the salt. After the gradual precipitation of each fraction over a period of about 15 min * A preliminary report of this work has been published (1). This work has been supported by grantsinaid from the National Cancer nstitute, National nstitutes of Health, United States Public Health Service, the Damon Runyon Memorial Fund for Cancer Research, nc., and the National Science Foundation. t United States Public Health Service Research Fellow of the National nstitute of Neurological Diseases and Blindness (195455). 1019

2 1020 BOSYNTHESS OF PURNES. X utes, the suspension was stirred for 10 minutes prior to removal of precipitated protein by centrifugation. n this manner, three separat,e protein fractions were obtained: Fractions,., and, precipitating from 0 to 35, 35 to 45, and 45 to 60 per cent saturation of ammonium sulfate, respectively. Fraction, was discarded. For preparative purposes Fraction was taken up in 0.05 M sodium phosphate buffer, ph 7.4, and the concentration of protein was adjusted to approximately 20 mg. per ml. by determination of optical density at 280 rnp. This material could be stored for 3 to 4 weeks at 2O with little loss of enzymatic activity. For studies concerned with the mechanism of the reaction, however, Fraction from the above procedure was suspended in 25 ml. of water and permitted to stand overnight at 0 in order to effect fine dispersion of the insoluble material and to obtain proper solution of desired enzymes. The insoluble material was removed by centrifugation and the residue was washed with 5 ml. of water. This extract was then diluted to 84 ml. with water and adjusted to a final concentration of N with potassium acetate buffer, ph 6.0. This solution which contained 530 mg. of protein was then treated with 25 ml. of alumina Cy (12.3 mg. per ml.) (4). The suspension was stirred at 0 for 10 minutes and the insoluble material was removed by centrifugation and discarded. The supernatant solution contamed the active enzyme at a protein concentration of 1.16 mg. per ml. n this last step the enzyme was purified approximately threefold and an enzyme contaminant was removed which catalyzed the conversion of glutamic acid to glutamine. Fraction, which was used for the enzymatic assay of FGAM by measurement of the conversion of FGAM to AR, was dissolved in water and the protein concentration of this solution was adjusted to approximately 22 mg. per ml. The enzyme remained active for 6 weeks or more at 2 in this solution. EXPERMENTAL Sequence of Enzymatic Action and Requirement for SubstratesNeither Fraction nor Fraction separately had the ability to effect the conversion of FGAR to AR, but, when combined, produced excellent synthesis of the a&mine. The sequence of action of these two fractions was readily determined by double incubation experiments which are illustrated in Table (Vessels 1 and 2). These experiments demonstrated the forma tion of a. heatstable intermediate (FGAM) in the presence of Fraction, which was subsequently converted to AR upon the addition of Fraction. 1 The extinction coefficient of 1 mg. of protein at 280 rnp in a cell with a 1 cm. light path is 1.6.

3 B. LEVENBERG AND J. M. BUCHANAN 1021 The substrate requirements for the individual steps of the synthesis are likewise shown in Table. As may be seen, glutamine, ATP, and FGAR are all required together with the enzymes of Fraction for the synthesis of the intermediate (FGAM). Although it was not possible to show it in this experiment, ATP was essential for the reactions catalyzed by both Fraction and Fraction. When the experiment was carried out with an ammonium sulfate fraction as a source of Fraction, it was found that Vessel No. TABLE Sequence of Enzymatic Action of Fractions and ; Substrate Requirements for Synthesis of FGAM frm FGAR ElUpe Fraction 1st incubation Substrates T FGAR T added ATP $$, Enzyme Fraction 2nd FGAR incubation Substrates ATP added T _ Glutamine AR synthesized n the first incubation each vessel contained in a final volume of 0.43 ml. the following materials when indicated: 0.08 pmole of the barium salt of FGAR, 6 pmoles of Lglutamine, and 1.2 pmoles of disodium ATP, 14 pmoles of sodium phosphate buffer, ph 7.4, 10 pmoles of KzSO~, 3 rmoles of MgSOd, and 0.05 ml. of either Fraction or Fraction (protein concentration 15 mg. per ml.). Time of first incubation, 30 minutes at 38. Reactions stopped by heating vessels in a water bath at 100 for 40 seconds. The vessels were then chilled in an ice bath and centrifuged, but the supernatant solution was not removed from the sediment. The indicated substrates or enzymes were then added to the vessels, the final volume was made up to 0.62 ml. with water and the incubation was continued at 38 for 30 minutes. The vessels were chilled in ice and the reaction was stopped by the addition of 0.1 ml. of 30 per cent trichloroacetic acid. After centrifugation, an aliquot of 0.04 ml. was removed for the determination of AR as described in Paper X (2). glutamic acid but not aspartic acid or asparagine could partially substitute for glutamine. However, when Fraction was further purified on alumina Cy as specified above, and used in the reaction, glutamine was specifically required and glutamic acid had no activity as a substrate. The enzymatic synthesis of FGAM on a relatively large scale and its isolation in pure form are presented below. Enzymatic Synthesis and solation of FGAM ncubation ProcedureEach vessel contained, in a final volume of 50 ml., the following quantities of materials expressed in micromoles: barium

4 1022 BOSYNTHES9 OF PUBNEB. X FGAR1C 15, nglutamine 68, disodium ATP 8.7, sodium phosphate buffer, ph 7.4, 215, potassium sulfate 230, magnesium sulfate 65, and 90 mg. (4.5 ml.) of Fraction. Four such vessels were incubated for 15 minutes at 38, and the reaction mixtures were then pooled in one large flask. The contents of the flask were heated in a water bath at 100 for 4 minutes and then chilled in an ice bath. The precipitated protein was removed by centrifugation. Enzymatic assay of the FGAM synthesized indicated a yield of approximately Analysis* TABLE Chemical Anulysfs of (anfor@)glycinomidins pmoles Ba salt of FGAM sample B Sample b per mg. Ribotide Glycine.... Pentose... Formate norganic P Total P Organic P A&dlabile N Total N C" Seoondary phosphate hydrogen ion * The listed compounds were obtained upon hydrolysis or combustion. Their analyses are described in a previous paper (5) mg. of the Ba salt of FGAM is equivalent to 2.22 pmoles. pnwles p3 mg.. Molecular ratio (glycine 1.00) Sample 8 35 per cent, based upon the quantity of FGAR added to the incubation system. solation an& Purification of Barium Salt of FGAlWThe ion exchange chromatographic procedures employed for the isolation of FGAM, as well aa the method of preparation of both the crude and purified barium salts of this compound, differed little from those developed in the case of AR (2). All operations were again carried out at 3, although the stability of FGAM is believed to be much greater than that of the arylamine ribotide. Two minor alterations in the procedure were made: (1) the deproteinized incubation mixture was placed in equal portions on twentythree columns of Dowex,l acetate (0.8 X 14 cm.), and (2) FGAM was eluted with 0.03 M ammonium acetate buffer, ph 5.3, from these columns. n the final purification step in which a single column was employed, the elut _ Sample b

5 B. LEVENBERQ AND J. M. BUCHANAN 1023 ing solution was 0.01 M ammonium acetate buffer, ph 5.3. n order to determine the fractions of the chromatogram in which FGAM was present, an aliquot of each solution was removed, evaporated to dryness under an infrared lamp on a copper planchet, and assayed for radioactivity. t is O PH FO. 1. Titration curve of (ornformyl)glycinamidine ribotide mg. of the barium salt of Wlabeled FGAM were passed through a Dowex 50 (H) column (0.3 X 1.3 cm.) which was then washed with 3 ml. of HO. The total material which passed through the column contained the equivalent of 2.00 mg. of material. This was titrated with a glass electrode with N NaOH. H2C fh CHO FG. 2. Structure of (crvformyl)glycinamidine ribotide of interest that a small portion of the radioactivity eluted from the column employed in the final purification step was present in a compound which behaved chromatographically and enzymatically identically with FGAR. Since all of the unchanged FGAR was separated from FGAM during the first chromatographic step, it was concluded that a small amount of FGAM was hydrolyzed to FGAR during purification.

6 1024 BOSYNTHESS OF PURNES. X The purified barium salt of FGAM was dried in vucuo over PzOs prior to analysis. t was obtained in an overall yield of 11 per cent (based upon the quantity of FGAR present in the incubation). Analysis and Chemical Properties of FGAMThe data obtained on chemical analysis of the components liberated upon hydrolysis of two different samples of the barium salt of FGAM are presented in Table. The methods of calculation are the same as those used for the analyses of AR in Table of Paper X. t is seen that FGAM is composed of moieties which yield, upon hydrolysis or combustion, glycine, formate, pentose, organic phosphate, acidlabile nitrogen, and total nitrogen in the same approximate molecular ratios as those of AR, namely, 1: 1: 1: 1:2: 3, respectively. TABLE Adenine Nucleotide Requirement for Conversion of FGAM to AR Nucleotide added to basic system* AR synthesized wipmoles None... 0 AMP ADP ATP , * The basic system consisted of 0.055pmole of the barium salt of FGAM, 10 rmoles of sodium phosphate buffer, ph 7.4, 10 rmoles of KtSOa, 3.5 pmoles of MgS04, and 0.1 ml. of the enzyme, Fraction. Either adenosine5 phosphate (AMPB ), ADP, or ATP added as indicated at a level of 1 rmole. Total volume, 0.5 ml. The vessels were incubated for 30 minutes at 38. The extremely weak affinity of FGAM for the anion exchange resin indicated the presence within the molecule of a rather basic constituent. This belief was further substantiated by the results of an electrometric titration of FGAM (Sample b), which is shown in Fig. 1. Approximately 2 equivalents of Hf ion were titrated per mole of compound between ph 4 and 10. One of the equivalents corresponded to the dissociation of a secondary hydrogen ion of phosphate at pk 6.0 (see also Table ) and the other to the dissociation of a hydrogen ion of a more basic group at approximately pk 9.2. Owing to the fact that the second group was titrated partially at a ph at which the accuracy of the glass electrode began to diminish, it was not possible to obtain the exact number of equivalents titrated per mg. of material. An estimation of the pk value of the titration was made, however, by measuring the inflection point of the curve. Since the aamino nitrogen of FGAM is substituted by a formyl residue, it was concluded that the group dissociating at pk 9.2 was an amidine

7 B. LEVENBERG AND J. M. BUCHANAN 1025 which resulted from the transfer of the amide nitrogen of glutamine to FGAR. Hydrolysis of FGAM by acid (1 N HCl at 100 for 1 hour) resulted in the liberation of glycine as the only ninhydrinreactive spot detectable on paper chromatograms (solvent system = 95 per cent ethanolconcentrated NH,OH, 95 : 5). No reaction was obtained for FGAM in either the Pauly diazo reaction ATP R5P ATP CH2NH2 GLUTAhWE GYCM J PRPP O&NH LRP CH2NH ATP CH2 CHO GLUTAMNE,&NH c HZNH hp GM ACTVE MP HCOOH ) AKAR NH HCN\ CHO ATP 3 P, c N LRP H2N LRP FGAR FGAM AR H2N\p0 3 c ACTVE CO,, ATP HCOOH CN cn\ 4 < 2 11 & ASPARK ACD /c \ /CN H N N 2 RP AlGAR gg FG. 3. An abbreviated scheme for the biosynthesis of inosinic acid de nouo. The double arrows in certain of the reactions indicate that preliminary evidence has been obtained for the existence of additional intermediates. RP refers to ribosed phosphate (as a ribotide), R5P to ribosedphosphate, and ACAR to 5amino4imidazolecarboxamide ribotide. The other abbreviations are defined in the text. (6) or the BrattonMarshall test. Similar to FGAR (5), FGAM exhibits only weak end absorption below 240 mp. The above chemical analyses and properties of this compound, together with the metabolic experiments reported below, permit the formulation of the structure of FGAM as shown in Fig. 2. Metabolism of FGAMFGAM was tested for its ability to function as an intermediate in the biosynthesis of inosinic acid (MP). n the presence of the 13 to 33 ethanol fraction of pigeon liver extract it was found that the radioactivity of FGAMlC * was converted to MP and that the specific activity of the newly formed MP was not affected by the inclusion of a large bank of unlabeled glycine in the incubation mixture. Upon isolation of FGAM in pure form it was possible to study more )CH LRP

8 1026 BOSYNTHESS OF PURNES. X specifically the substrates required for its conversion to AR. As shown in Table, formation of the imidazole ring occurred only upon addition of one of the adenine nucleotides, ATP or adenosine diphosphate (ADP), to these relatively impure enzyme preparations. Since ATP was more effective at equivalent concentrations than ADP, it is probable that ATP is the actual substrate. DSCUSSON The results reported in this paper demonstrate that the imidazole ring of the purines is formed enzymatically only after the introduction of what would correspond to nitrogen atom 3 of the purine molecule. t is of interest, furthermore, that ATP is required in the enzymatic step in which ring closure occurs. These studies permit the presentation of a scheme of reactions, which, although not yet complete, account for the major steps of purine biosynthesis (Fig. 3). The double arrows in certain of the reactions indicate that evidence has been obtained for the existence of new intermediates not shown in the scheme. n Step 1, 5phosphoribosyl pyrophosphate (PRPP), ATP, and glutamine are compounds shown to be involved in the synthesis of GAR from glycine (7, 8). A recent report (8) has implicated 5phosphoribosylamine as the ribose compound which reacts with glycine and ATP to form glycinamide ribotide (GAR), but it has not yet been possible to isolate thii compound as a result of an enzymatic reaction between PRPP and glutamine. GAR may be directly formylated by formate in the presence of ATP and a suitable folic acid compound (9) or it may undergo transformylation with inosinic acid to yield FGAR (10). The conversion of FGAR to FGAM and then to AR has been reported in this and Paper X. Lukens and Buchanan (11) in this laboratory have now isolated a new intermediate involved in the conversion of AR to 5amino4imidazolecarboxamide ribotide. n a further step, Warren and Flaks (12) have implicated 5 formamido4~imidazolecarboxamide ribotide in the conversion of 5amino &imidazolecarboxamide ribotide to inosinic acid. t will be noted that, almost without exception, the purine ring is formed by the withdrawal of the elements of water in a series of enzymatic reactions in which precursors are utilized stepwise for the formation of intermediates which are transformed eventually into inosinic acid. SUMMARY Fractionation of the enzyme system of pigeon liver concerned with the conversion of (anformyl)glycinamide ribotide to 5aminoimidazole ribotide has permitted the accumulation of a new compound which is an inter

9 B. LEVENBERG AND J. M. BUCHANAN 1027 mediate of this reaction and of purine biosynthesis de novo. This compound has been isolated in pure form and identified as (arnformyl)glycinamidine ribotide. BBLOGRAPHY 1. Buchanan, J. M., Levenberg, B., and Lukens, L. N., Abstracts, American Chemical Society, 128th meeting, Minneapolis, 12C (1955). 2. Levenberg, B., and Buchanan, J. M., J. Biol. Chem., 334, 1005 (1957). 3. Bratton, A. C., and Marshall, E. K., Jr., J. Biol. Chem., 128,537 (1939). 4. Colowick, S. P., in Colowick, S. P., and Kaplan, N. O., Methods in enzymology, New York, 1, 90 (1955). 5. Hartman, S. C., Levenberg, B., and Buchanan, J. M., J. Biol. Chem., 221, 1057 (1956). 6. Koessler, K. K., and Hanke, M. T., J. BioZ. Chem., 39, 497 (1919). 7. Hartman, S. C., Levenberg, B., and Buchanan, J. M., J. Am. Chem. Sot., 77,501 (1955). 8. Goldthwait, D. A., Greenberg, G. R., and Peabody, R. A., Biochim. et biophys. acta, 18, 148 (1955). 9. Goldthwait, D. A., Peabody, R. A., and Greenberg, G. R., J. Am. Chem. Sot., 76, 5258 (1954). 10. Levenberg, B., Hartman, S. C., and Buchanan, J. M., Federation Proc., 14, 243 (1955). 11. Lukens, L. N., and Buchanan, J. M., Federation Proc., 16, 306 (1956). 12. Warren, L., and Flaks, J. G., Federation Proc., 16, 379 (1956).

10 BOSYNTHESS OF THE PURNES: X. STRUCTURE, ENZYMATC SYNTHESS, AND METABOLSM OF ( NFORMYL)GLYCNAMDNE α RBOTDE Bruce Levenberg and John M. Buchanan J. Biol. Chem. 1957, 224: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at html#reflist1

Enzymatic Assay of ACTOMYOSIN ATPase Assay

Enzymatic Assay of ACTOMYOSIN PRINCIPLE: ATP + H 2 O ATPase > ADP + P i Abbreviations used: ATP = Adenosine 5'-Triphosphate ATPase = Adenosine 5'-Triphosphatase ADP = Adenosine 5'-Diphosphate P i = Inorganic