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7.11: Purine de novo Biosynthesis - Biology

7.11: Purine de novo Biosynthesis - Biology


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PRPP amidotransferase is regulated partly by GMP and partly by AMP. The presence of either of these can reduce the enzyme’s activity. Only when both are present is the enzyme fully inactivated. Subsequent reactions include adding glycine, adding carbon (from N 10-formyltetrahydrofolate), adding amine (from glutamine), closing of the first ring, addition of carboxyl (from ( ext{CO}_2)), addition of aspartate, loss of fumarate (a net gain of an amine), addition of another carbon (from ( ext{N}_10)-formyltetrahydrofolate), and closing of the second ring to form inosine monophosphate (IMP).


IMP is a branch point for the synthesis of the adenine and guanine nucleotides. The pathway leading from IMP to AMP involves addition of amine from asparate and requires energy from GTP. The pathway from IMP to GMP involves an oxidation and addition of an amine from glutamine. It also requires energy from ATP. The pathway leading to GMP is inhibited by its end product and the pathway to AMP is inhibited by its end product.


Thus, balance of the purine nucleotides is achieved from the IMP branch point forward. It is at this point that the significance of the unusual regulation of PRPP amidotransferase becomes apparent. If there is an imbalance of AMP or GMP, the enzyme is slowed, but not stopped, thus allowing the reactions leading to IMP to proceed, albeit slowly. At IMP, the nucleotide in excess feedback inhibits its own synthesis, thus allowing the partner purine nucleotide to be made and balance to be achieved. When both nucleotides are in abundance, then PRPP amidotransferase is fully inhibited and the production of purines is stopped, thus preventing them from over-accumulating.


AMPK Activation via Modulation of De Novo Purine Biosynthesis with an Inhibitor of ATIC Homodimerization

5-Aminoimidazole-4-carboxamide ribonucleotide (known as ZMP) is a metabolite produced in de novo purine biosynthesis and histidine biosynthesis, but only utilized in the cell by a homodimeric bifunctional enzyme (called ATIC) that catalyzes the last two steps of de novo purine biosynthesis. ZMP is known to act as an allosteric activator of the cellular energy sensor adenosine monophosphate-activated protein kinase (AMPK), when exogenously administered as the corresponding cell-permeable ribonucleoside. Here, we demonstrate that endogenous ZMP, produced by the aforementioned metabolic pathways, is also capable of activating AMPK. Using an inhibitor of ATIC homodimerization to block the ninth step of de novo purine biosynthesis, we demonstrate that the subsequent increase in endogenous ZMP activates AMPK and its downstream signaling pathways. We go on to illustrate the viability of using this approach to AMPK activation as a therapeutic strategy with an in vivo mouse model for metabolic disorders.

Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.


7.11: Purine de novo Biosynthesis - Biology

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Ribose-5-Phosphate to IMP synthesis

Step 1: Amination

The starting material for purine biosynthesis is Ribose-5-P, a product of the Hexose MonoPhosphate Shunt or Pentose Phosphate pathway (HMP Shunt). The ribose-5-P is converted into phosphoribosyl pyrophosphate by Pyrophospho Kinase in this reaction ATP is consumed.

Step 2: Addition of N9

One nitrogen is added on Ribose-5-P, to form 5-phosphoribosyl-1-amine (PRA). The nitrogen is donated by Glutamine. Ribose-5-Phosphate is derived from PRPP.

The reaction needs energy from ATP hydrolysis. This is the rate-limiting enzyme of this pathway. This step is inhibited by azaserine, the anticancer drug.

Step 3: Incorporation of C4, C5, and N7

The phosphoribosyl amine (PRA) is condensed with glycine it forms Glycinamide ribotide (GAR). This reaction is catalyzed by GAR Synthase.

Step 4: Adition Of C8

Glycinamide ribotide is converted into a Formyl glycine amide ribotide (FGAR). This reaction is catalyzed by transformylase. Here Formyl donor is N 10 -Formyl-THF.

Step 5: Addition of N3

Formyl Glycine ribotide is converted into Formylglycinaidine ribotide (FGAM) in the presence of the enzyme FGAM synthetase. Here amide donor is Glutamine and it is ATP consumed reaction. This enzyme is also inhibited by azaserine.

Step 6: Cyclisation (Closure of Ring)

FGAM is converted into 5-amino imidazole ribotide (AIR). This reaction is catalyzed by AIR Synthetase. Here ATP is consuming.

Step 7: Addition of C6

Amino imidazole ribotide is converted into 5-amino-4-Carboxy-Amino-Imidazole Ribonucleotide (CAIR). This reaction catalyzed by AIR carboxylase. In this reaction, Carbonic acid is substituted on a 4 th carbon atom as in the form of the Carboxyl group (CAIR).

This carbon dioxide fixation reaction does not require biotin or ATP.

Step 8: Addition of N1

Carboxy Amino Imidazole has converted into 5-AminoImidazole (N-Succinylocarboxamide) ribotide (SACAIR). This reaction is catalyzed by SACAIR synthetase. In this reaction, one Aspartic acid linked with Carboxyl group ATP is consumed.

Step 9: Removal of Fumaric acid

SACAIR is converted into 5-AminoImidazole-4-CarboxyAmide Ribotide (AICAR). This reaction is catalyzed by Adenosuccinate Lyase. The linked Aspartic acid hydrolyzed as Fumarate, which directly enters into TCA cycle.

The amino group of aspartic acid becomes the first nitrogen of the purine ring.

Step 10: Addition of C2

AICAR is converted into 5-FormaminoImidazole-4-Carboxamide Ribotide (FAICAR). This reaction is catalyzed by Transformylase. Here formyl group donor is N 10 -Formyl THF. This carbon is derived from the one-carbon pool. In folic acid deficiency, this step is blocked hence orange-colored FAICAR is excreted in the urine.

Step 11: Cyclization

FAICAR is converted into Inosine Mono Phosphate (IMP) by the catalyzation process. This reaction is catalyzed by IMP Cyclohydrolase. It contains the purine, hypoxanthine.

Donor atomAdded featuresSpecialProduct
GlutamineN9Rate-limitingPRA
GlycineC4, C5 and N7ATP requiredGAR
Methenyl-THFAC8FGAR
GlutamineN3ATP requiredFGAM
Ring closureATP needAIR
Carbon dioxideC6ACAIR
Aspartic acidN1 ATP requiredSAICAR
Fumarate removedAICAR
Formyl THFAC2FAICAR
Ring closureIMP


Biosynthesis of Purine Nucleotides, Pyrimidine Nucleotides and Deoxyribonucleotides

De novo (all over again) synthesis of purine nucleotides is synthesis of purines anew. The purine ring is synthesized along with the nucleotide i.e. attached to the ribose sugar provided from HMP pathway. This pathway supplies ribose sugar for the formation of the nucleotide. Activated form of D-ribose-5-phosphate serves as the starting material on which purine ring is build up step by step.

Precursors of the members of purine ring are:

i. N-1 is contributed by nitrogen of aspartate.

ii. N-3 and N-9 arise from amide nitrogen of glutamine.

iii. C-2 and C-8 originate from the formate.

iv. C-6 is embedded from respiratory carbon dioxide.

v. C-4, C-5 and N-7 are taken up from glycine.

Regulation of purine nucleotide biosynthesis:

Purine biosynthesis is regulated by feedback inhibition. This inhibition is in the 1 st step. It is the committed step which is generally irreversible. Once the committed step is passed over, the product has to be formed.

The different mechanisms by which it is regulated are:

Salvage Pathway:

The de-novo synthesis does not occur in all the cells. Brain cells and leukocytes lack this mechanism. In these cells purine synthesis occurs by salvage pathway. Salvage pathway involves synthesis of purine nucleotides from free purine bases, which are salvaged from dietary sources and tissue breakdown. This pathway is promoted by the action of two enzymes which convert free purines into purine nucleotides for reuse.

The enzymes are:

(1) Adenine phosphoribosyl transferase and

(2) Hypoxanthine guanine phosphoribosyl transferase (HGPRT).

This is a genetic disorder caused due to the deficiency of the enzyme ‘Hypoxanthine Guanine Phospho Ribosyl Transferase (HGPRT)’. When this enzyme is deficient, guanine, xanthine and hypoxanthine are not salvaged and hence degraded to uric acid. This is especially seen in male children. In female children the gene is recessive and is a carrier. It is a male dominant gene. Such males show (1) mental retardation and (2) tendency for self-destruction.

Biosynthesis of Pyrimidine Nucleotides:

Pyrimidine nucleotide biosynthesis takes place in a different manner from that of purine nucleotides. The six membered pyrimidine ring is made first and then attached to ribose phosphate. The synthesis begins with carbon dioxide and ammonia combining to form carbamoyl phosphate catalysed by the cytosolic enzyme carbamoyl phosphate synthetase-II.

Carbamoyl phosphate combines with aspartate to form carbamoyl aspartate aided by the enzyme aspartate transcarbamoylase. Dihydroorotate is formed from carbamoyl aspartate by removal of water and closure of the ring under the influence of the enzyme dihydroorotase.

Dihydroorotase is oxidized to orotic acid by dehydrogenase which uses NAD + as the electron acceptor. Orotic acid is attached to ribose to yield orotidylic acid. Orotidylate is then decaroxylated to form uridylate. Uridylate is then converted to all the other pyrimidine nucleotides viz., CMP, UMP & TMP. The reaction steps involved in the biosynthesis of pyrimidine nucleotides are given under.

Regulation of Pyrimidine Biosynthesis:

Regulation of pyrimidine biosynthesis is by feed back inhibition at the committed step i.e. the reaction catalysed by the enzyme aspartate transcarbamoylase. This is negatively inhibited by the end product i.e. CTP. The second site is at carbamoyl phosphate synthase- II which is feedback inhibited by UMP.

Orotic Aciduria:

It is a metabolic disorder of pyrimidine biosynthesis characterized by accumulation of orotic acid in blood and its increased excretion in urine. It is caused due to the deficiency of enzyme orotidylic acid phosphorylase and orotidylic acid decarboxylase or orotic phosphoribosyl transferase. This leads to non-conversion of orotic acid to UMP. This may even affect the synthesis of other nucleotides. It is generally found in children who show retarded mental development and growth as there is no proper synthesis of DNA. They show megaloblastic anemia. This can be overcome by injection of CTP and UTP.

Biosynthesis of Deoxyribonucleotides:

Deoxyribonucleotides are obtained from ribonucleotides. Thioredoxin is a protein which takes part in the conversion of ribonucleotides to deoxyribonucleotides.


Quantitative analysis of purine nucleotides indicates that purinosomes increase de novo purine biosynthesis

Enzymes in the de novo purine biosynthesis pathway are recruited to form a dynamic metabolic complex referred to as the purinosome. Previous studies have demonstrated that purinosome assembly responds to purine levels in culture medium. Purine-depleted medium or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) treatment stimulates the purinosome assembly in HeLa cells. Here, several metabolomic technologies were applied to quantify the static cellular levels of purine nucleotides and measure the de novo biosynthesis rate of IMP, AMP, and GMP. Direct comparison of purinosome-rich cells (cultured in purine-depleted medium) and normal cells showed a 3-fold increase in IMP concentration in purinosome-rich cells and similar levels of AMP, GMP, and ratios of AMP/GMP and ATP/ADP for both. In addition, a higher level of IMP was also observed in HeLa cells treated with DMAT. Furthermore, increases in the de novo IMP/AMP/GMP biosynthetic flux rate under purine-depleted condition were observed. The synthetic enzymes, adenylosuccinate synthase (ADSS) and inosine monophosphate dehydrogenase (IMPDH), downstream of IMP were also shown to be part of the purinosome. Collectively, these results provide further evidence that purinosome assembly is directly related to activated de novo purine biosynthesis, consistent with the functionality of the purinosome.

Keywords: Mass Spectrometry (MS) Metabolic Flux Metabolomics Nucleoside/Nucleotide Biosynthesis Protein Complex Purine Purinosome.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.


7.11: Purine de novo Biosynthesis - Biology

Synonyms: purine biosynthesis 2

Purine nucleotides participate in many aspects of cellular metabolism including the structure of DNA and RNA, serving as enzyme cofactors, functioning in cellular signaling, acting as phosphate group donors, and generating cellular energy [Carter08]. Maintenance of the proper balance of intracellular pools of purine nucleotides is critical to normal function [Zhang08c]. This occurs through a combination of de novo purine biosynthesis and superpathway of purine nucleotide salvage [Zhao13]. The de novo purine biosynthesis is more energy consuming than the purine salvage pathway.

The de novo biosynthetic pathway for purine nucleotides is highly conserved among organisms, but its regulation and organization of the genes encoding the enzymes vary. The pathway forms IMP from PRPP and glutamine using several molecules of ATP in ten enzymatic reactions [Ng10]. The reactions of this pathway are catalyzed by ten separate enzymes in most prokaryotes [Senecoff96]. However, in humans the ten highly conserved steps leading to IMP synthesis are catalyzed by just six enzymes, including one trifunctional and two bifunctional enzymes.

This pathway shows the additional steps downstream of IMP leading to the formation of GTP, dGTP, ATP and dATP.

Fluorescent microscopy studies on cancer cells has shown that the six proteins of the pathway leading upto the formation of IMP, assemble in a functional multienzyme complex termed the "purinosome", within the cytoplasm [An08]. This complex is dynamically reversible [Deng12]. Further investigation of the purinosome has shed light on some of the accessory proteins that may aid purinosome assembly and hence de novo purine biosynthesis, which include protein kinases and heat shock proteins Hsp70 and Hsp90 [An10][French13].

The cytosolic purinosome clusters associate with microtubule filaments not connected with the actin network. Nocodazole, an inhibitor of microtubule formation, disrupted purinosomes formation and decreased flux through the de novo purine biosynthetic pathway [An10a].

Rapidly dividing cells favor the de novo purine biosynthetic pathway, and it has long been considered an ideal target for anticancer, antiviral, and antimicrobial chemotherapeutics. Folates are required cofactors for many of the enzymes of purine biosynthesis, and anti-folates have also gained widespread use as chemotherapeutic agents to disrupt the pathway [Zhao13].


Cloning of three human multifunctional de novo purine biosynthetic genes by functional complementation of yeast mutations.

Functional complementation of mutations in the yeast Saccharomyces cerevisiae has been used to clone three multifunctional human genes involved in de novo purine biosynthesis. A HepG2 cDNA library constructed in a yeast expression vector was used to transform yeast strains with mutations in adenine biosynthetic genes. Clones were isolated that complement mutations in the yeast ADE2, ADE3, and ADE8 genes. The cDNA that complemented the ade8 (phosphoribosylglycinamide formyltransferase, GART) mutation, also complemented the ade5 (phosphoribosylglycinamide synthetase) and ade7 [phosphoribosylaminoimidazole synthetase (AIRS also known as PAIS)] mutations, indicating that it is the human trifunctional GART gene. Supporting data include homology between the AIRS and GART domains of this gene and the published sequence of these domains from other organisms, and localization of the cloned gene to human chromosome 21, where the GART gene has been shown to map. The cDNA that complemented ade2 (phosphoribosylaminoimidazole carboxylase) also complemented ade1 (phosphoribosylaminoimidazole succinocarboxamide synthetase), supporting earlier data suggesting that in some organisms these functions are part of a bifunctional protein. The cDNA that complemented ade3 (formyltetrahydrofolate synthetase) is different from the recently isolated human cDNA encoding this enzyme and instead appears to encode a related mitochondrial enzyme.


Materials and methods

Chemicals

Isotopically labeled glycine (U- 13 C2, 15 N) was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). AICAR, 5-aminoimidazole-4-carboxamide riboside (AICAr) and adenylosuccinic acid (SAMP) were purchased from Toronto Research Chemicals Inc. (North York, Canada). SAICAR, succinylaminoimidazolecarboxamide riboside (SAICAr), succinyladenosine (SAdo), AIR, 5-aminoimidazole riboside (AIr), CAIR, carboxyaminoimidazole riboside (CAIr), and N 10 -formyl-tetrahydrofolate (N 10 -formyl-THF) were prepared as previously described [11, 14]. Calf intestinal alkaline phosphatase (CIP) and NEB3 buffer were purchased from New England Biolabs (NEB, Ipswich, MA, USA), and Dulbecco's minimum essential medium (DMEM), F12 nutrient mix, and fetal bovine serum (FBS) were obtained from Life Technologies, ThermoFisher Scientific (MA, USA). Minimum Essential Medium (MEM) was obtained from BioSera (Nuaille, France). All other chemicals were purchased from Sigma-Aldrich (St. Louis, USA).

Preparation and purification of substrates

GAR, FGAR, and FGAMR intermediates were synthesized biochemically using bacterial recombinant enzymes. FAICAR was prepared by inorganic synthesis. We did not attempt to synthesize PRA, as it is known to be unstable in vivo [15, 16].

The initial concentration of all compounds ranged from 57 μM in samples of FGAMR/r to 124 μM in samples of GAR/r (see S1 Table).

The bacterial recombinant enzymes phosphoribosylglycinamide synthetase (GARS) and phosphoribosylglycinamide formyltransferase (GARTF) were expressed and purified as fused protein maltose binding protein MBP-GARS and MBP-GARTF using the pMAL TM Protein Fusion and Purification System (New England Biolabs Inc., USA), as described previously [17].

To produce recombinant bacterial phosphoribosylformylglycinamide synthetase fused with a C-terminal polyhistidine tag (6H-PurL), the gene was introduced into the p6H vector, expressed in Escherichia coli, and purified on a Co 2+ -immobilized metal affinity chromatography column (GE Healthcare) according to standard procedure.

GAR/r preparation.

The reaction mixture containing 5.7 mM ribose-5-phosphate, 0.7 mM ATP, 10 mM glycine, 10 mM ammonium hydroxide, 12.7 mM magnesium chloride, 20 mM phosphate buffer pH 7.4, and 0.4 μg/μL purified MBP-GARS was incubated at 37°C for four hours. The reaction was analyzed by high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). The riboside form was prepared by dephosphorylation with 1 U of CIP from NEB at 37°C for four hours.

FGAR/r preparation.

The reaction mixture containing 5.7 mM ribose-5-phosphate, 0.7 mM ATP, 10 mM glycine, 10 mM ammonium hydroxide, 12.7 mM magnesium chloride, 0.1 mM N 10 -formyl-THF, 20 mM phosphate buffer pH 7.4, 0.4 μg/μL MBP-GARS, and 0.4 μg/μL MBP-GARTF was incubated at 37°C for four hours. The subsequent procedure was the same as in GAR/r preparation.

FGAMR/r preparation.

A total of 200 μL of the reaction mixture from the synthesis of FGAR was incubated with 2 mM glutamine, 2 mM ATP, and 0.25 μg/μL of purified 6H-PurL at 37°C for four hours. The subsequent procedure was the same as in GAR/r preparation.

FAICAR/r preparation.

FAICAr was prepared according to Lukens et al. [18]. FAICAR was prepared by adjusting the procedure used for the synthesis of FAICAr. In brief, we incubated 10 mg of AICAR with 11 mg of NaOH, 136 μL of formic acid and 250 μL of acetic anhydride for 1 hour at 37°C. The product of the reaction was analyzed by HPLC-MS.

Cell cultivation and harvesting

We used the CRISPR-Cas9 genome-edited HeLa cells CR-GART, CR-PFAS, CR-PAICS, CR-ADSL, and CR-ATIC prepared by Baresova et al. in 2016 [11]. CR-HGPRT cells (hypoxanthine-guanine phosphoribosyltransferase deficient) were prepared analogously [19]. HeLa cells were cultured in a humidified atmosphere and incubated with 5% CO2 at 37°C. All cells (knockout and control) were maintained in DMEM/F12 nutrient mix medium supplemented with 10% FBS (Gibco, Invitrogen) and 1% penicillin/streptomycin. The medium of the knockout cells was enriched with 3x10 -5 M of adenine. Twenty-four hours prior to the experiment, all the cell types were cultivated in purine-depleted DMEM supplemented with dialyzed 10% FBS [11] and 1% penicillin/streptomycin. Two hours prior to cell harvesting, the cells were washed with PBS and placed into 5 mL of glycine-free MEM with 500 μM of isotopically labeled glycine (U- 13 C2, 15 N) added. Each deficient cell line was cultivated in hexaplicate in 75-cm 2 flasks (approx. 5 million cells).

Cells were harvested by means of the modified quenching method described by Wojtowicz et al. [20]. Initially, the medium was transferred into a 15-mL plastic tube for subsequent analytical preparation. Cellular metabolism was quenched by spraying 40 mL of 60% aqueous cold methanol (v/v, -50°C) by means of a plastic syringe with a needle. The culture flasks were kept on ice and extracted with 1 mL of 80% methanol (v/v, -50°C), and the cells were mechanically detached using a cell scraper. The cell debris was drained out with a pipette. For an additional extraction, another 2 mL of cold methanol was added. The methanol extracts were combined, sonicated (30 s), and centrifuged (1800 g, 5 min, 4°C), and the supernatants were freeze-dried.

Preparation of cell lysates

A total of 500 μL of cold 80% methanol was added to each lyophilizate and thoroughly mixed. The samples were centrifuged at 15,000 g for 15 min at 4°C, and the supernatants were taken for analysis.

Preparation of cell media

The media were mixed using a vortex then, 100 μL of each sample was taken and 300 μL of 80% methanol was added. The samples were left at -80°C overnight. The extracts were centrifuged at 15,000 g for 15 min at 4°C, and the supernatants were analyzed.

Fragmentation analysis of PDNS intermediates and their dephosphorylated analogues (HPLC-HRMS n )

Chromatographic separation was achieved with hydrophilic interaction liquid chromatography using an Ultimate 3000 RS (ThermoFisher Scientific, MA, USA). The aminopropyl column (Luna NH2 3 μm 100 Å, 100 x 2 mm, Phenomenex, Torrance, USA) was maintained at 35°C. The mobile phase consisted of 20 mM ammonium acetate in water at pH 9.75 (mobile phase A) and acetonitrile (mobile phase B). The gradient elution was performed as follows: t = 0.0, 95% B t = 7.0–13.0, 10% B t = 14.0–17.0, 95% B. The flow rate was set to 0.3 mL/min, and the injection volume was 2 μL.

Multistage fragmentation analysis was performed on an Orbitrap Elite (ThermoFisher Scientific, MA, USA) operating in positive mode using electrospray ionization (capillary temperature 350°C, source heater temperature 300°C, sheath gas 10 arb. units, auxiliary gas 35 arb. units, sweep gas 0 arb. units). The electrospray voltage was set at +3.0 kV. Fragmentation for the most abundant fragments with intensities higher than 1E4 was performed using data-dependent analysis (DDA) or TreeRobot (HighChem, SK) otherwise, the selection of fragments was performed manually. Up to five of the most intense signals in MS 2 were isolated and further fragmented. Then, one to six of the most intense signals from each MS 3 spectrum were subjected to fragmentation to MS 4 . The subsequent MS n stages were also dependent on the intensities of the emerging fragments, usually producing spectra of the one or two most intense fragments from MS 4 /MS 5 . The fragmentation spectra were produced via collision-induced dissociation (CID) using 30 units of normalized collision energy the isolation width was 2 Da, and the injection time was 200 ms. All the fragmentation spectra were measured with a resolution of 120,000 full-width at half-maximum (FWHM) and with a mass error below 3 ppm. The multistage fragmentation spectra of each compound were organized into mass spectral trees. In every spectrum, the structures of the fragments belonging to the precursor (target) compound/fragment were identified with the predictive fragmentation software MassFrontier 7.0.5.09 SP3 (HighChem, SK).

Analysis of cell lysates and media

The chromatographic conditions were the same as in the HPLC-HRMS n analysis mentioned above. Detection was performed on an Orbitrap Elite operating in positive ionization mode with the same setting as above. The detection method was divided into four time segments. Full scan analysis within the mass range m/z 70–1000 was performed in the first (0.0–3.0 min) and fourth (12.0–17.0 min) segments. The selected ion monitoring (SIM) method was applied in the second segment (3.0–7.0 min) for the analysis of ribosides (m/z 177–417) and in the third segment (7.0–12.0 min) for the analysis of ribotides (m/z 257–497) to enhance the sensitivity towards these metabolites (except for the measurement of SAdo and SAMP, which had different m/z ranges: 379–389 and 459–469, respectively). The resolution was set to 60,000 FWHM. The mass error was below 3 ppm. All cell lines were measured in hexaplicate, and the intensity values are presented as averages. The identities of the accumulated compounds in both cell lysates and media were confirmed by MS 2 fragmentation analysis. Fragmentation spectra were produced via CID with the fragmentation energy set to 30 units of normalized collision energy.


Purines are biologically synthesized as nucleotides and in particular as ribotides, i.e. bases attached to ribose 5-phosphate. Both adenine and guanine are derived from the nucleotide inosine monophosphate (IMP), which is the first compound in the pathway to have a completely formed purine ring system.

IMP Edit

Inosine monophosphate is synthesized on a pre-existing ribose-phosphate through a complex pathway (as shown in the figure on the right). The source of the carbon and nitrogen atoms of the purine ring, 5 and 4 respectively, come from multiple sources. The amino acid glycine contributes all its carbon (2) and nitrogen (1) atoms, with additional nitrogen atoms from glutamine (2) and aspartic acid (1), and additional carbon atoms from formyl groups (2), which are transferred from the coenzyme tetrahydrofolate as 10-formyltetrahydrofolate, and a carbon atom from bicarbonate (1). Formyl groups build carbon-2 and carbon-8 in the purine ring system, which are the ones acting as bridges between two nitrogen atoms.

A key regulatory step is the production of 5-phospho-α-D-ribosyl 1-pyrophosphate (PRPP) by ribose phosphate pyrophosphokinase, which is activated by inorganic phosphate and inactivated by purine ribonucleotides. It is not the committed step to purine synthesis because PRPP is also used in pyrimidine synthesis and salvage pathways.

The first committed step is the reaction of PRPP, glutamine and water to 5'-phosphoribosylamine (PRA), glutamate, and pyrophosphate - catalyzed by amidophosphoribosyltransferase, which is activated by PRPP and inhibited by AMP, GMP and IMP.

PRPP + L-Glutamine + H2O → PRA + L-Glutamate + PPi

In the second step react PRA, glycine and ATP to create GAR, ADP, and pyrophosphate - catalyzed by phosphoribosylamine—glycine ligase (GAR synthetase). Due to the chemical lability of PRA, which has a half-life of 38 seconds at PH 7.5 and 37 °C, researchers have suggested that the compound is channeled from amidophosphoribosyltransferase to GAR synthetase in vivo. [1]

PRA + Glycine + ATP → GAR + ADP + Pi

fGAR + L-Glutamine + ATP → fGAM + L-Glutamate + ADP + Pi

fGAM + ATP → AIR + ADP + Pi + H2O

The eight is catalyzed by adenylosuccinate lyase.

The products AICAR and fumarate move on to two different pathways. AICAR serves as the reactant for the ninth step, while fumarate is transported to the citric acid cycle which can then skip the carbon dioxide evolution steps to produce malate. The conversion of fumarate to malate is catalyzed by fumarase. In this way, fumarate connects purine synthesis to the citric acid cycle. [2]

In eukaryotes the second, third, and fifth step are catalyzed by trifunctional purine biosynthetic protein adenosine-3, which is encoded by the GART gene.

Both ninth and tenth step are accomplished by a single protein named Bifunctional purine biosynthesis protein PURH, encoded by the ATIC gene.

GMP Edit

AMP Edit

Purines are metabolised by several enzymes:

Guanine Edit

  • A nuclease frees the nucleotide
  • A nucleotidase creates guanosine converts guanosine to guanine converts guanine to xanthine (a form of xanthine oxidoreductase) catalyzes the oxidation of xanthine to uric acid

Adenine Edit

  • A nuclease frees the nucleotide
    • A nucleotidase creates adenosine, then adenosine deaminase creates inosine
    • Alternatively, AMP deaminase creates inosinic acid, then a nucleotidase creates inosine

    Regulations of purine nucleotide biosynthesis Edit

    The formation of 5'-phosphoribosyalamine from glutamine and PRPP catalysed by PRPP amino transferase is the regulation point for purine synthesis. The enzyme is an allosteric enzyme, so it can be converted from IMP, GMP and AMP in high concentration binds the enzyme to exerts inhibition while PRPP is in large amount binds to the enzyme which causes activation. So IMP, GMP and AMP are inhibitors while PRPP is an activator. Between the formation of 5'-phosphoribosyl, aminoimidazole and IMP, there is no known regulation step.

    Purines from turnover of cellular nucleic acids (or from food) can also be salvaged and reused in new nucleotides.

    • The enzyme adenine phosphoribosyltransferase (APRT) salvages adenine.
    • The enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) salvages guanine and hypoxanthine. [3] (Genetic deficiency of HGPRT causes Lesch–Nyhan syndrome.)

    When a defective gene causes gaps to appear in the metabolic recycling process for purines and pyrimidines, these chemicals are not metabolised properly, and adults or children can suffer from any one of twenty-eight hereditary disorders, possibly some more as yet unknown. Symptoms can include gout, anaemia, epilepsy, delayed development, deafness, compulsive self-biting, kidney failure or stones, or loss of immunity.

    Purine metabolism can have imbalances that can arise from harmful nucleotide triphosphates incorporating into DNA and RNA which further lead to genetic disturbances and mutations, and as a result, give rise to several types of diseases. Some of the diseases are:

    1. Severe immunodeficiency by loss of adenosine deaminase.
    2. Hyperuricemia and Lesch–Nyhan syndrome by the loss of hypoxanthine-guanine phosphoribosyltransferase.
    3. Different types of cancer by an increase in the activities of enzymes like IMP dehydrogenase. [4]

    Modulation of purine metabolism has pharmacotherapeutic value.

    Purine synthesis inhibitors inhibit the proliferation of cells, especially leukocytes. These inhibitors include azathioprine, an immunosuppressant used in organ transplantation, autoimmune disease such as rheumatoid arthritis or inflammatory bowel disease such as Crohn's disease and ulcerative colitis.

    Mycophenolate mofetil is an immunosuppressant drug used to prevent rejection in organ transplantation it inhibits purine synthesis by blocking inositol monophosphate dehydrogenase. Also Methotrexate indirectly inhibits purine synthesis by blocking the metabolism of folic acid (it is an inhibitor of the dihydrofolate reductase).

    Allopurinol is a drug that inhibits the enzyme xanthine oxidoreductase and, thus, lowers the level of uric acid in the body. This may be useful in the treatment of gout, which is a disease caused by excess uric acid, forming crystals in joints.

    In order to understand how life arose, knowledge is required of the chemical pathways that permit formation of the key building blocks of life under plausible prebiotic conditions. Nam et al. [5] demonstrated the direct condensation of purine and pyrimidine nucleobases with ribose to give ribonucleosides in aqueous microdroplets, a key step leading to RNA formation. Also, a plausible prebiotic process for synthesizing purine ribonucleosides was presented by Becker et al. [6]


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