Step 2 Figure The anomeric carbon of the substrate PRPP is in the a-configuration; the product is a b-glycoside recall that all the biologically important nucleotides are b-glycosides.
The N atom of this N-glycoside becomes N-9 of the nine-membered purine ring; it is the first atom added in the construction of this ring.
Azaserine acts as an irreversible inhibitor of glutamine-dependent enzymes by covalently attaching to nucleophilic groups in the glutamine-binding site. The G series of nucleotides interacts at a guanine-specific allosteric site on the enzyme, whereas the adenine nucleotides act at an A-specific site. The pattern of inhibition by these nucleotides is competitive, thus ensuring that residual enzyme activity is expressed until sufficient amounts of both adenine and guanine nucleotides are synthesized.
Glutamine phosphoribosyl pyrophosphate amidotransferase is also sensitive to inhibition by the glutamine analog azaserine Figure Azaserine has been employed as an antitumor agent because it causes inactivation of glutamine-dependent enzymes in the purine biosynthetic pathway.
Step 3 is carried out by glycinamide ribonucleotide synthetase GAR synthetase via its ATP-dependent condensation of the glycine carboxyl group with the amine of 5-phosphoribosyl-b amine Figure The reaction proceeds in two stages. First, the glycine carboxyl group is activated via ATP-dependent phosphorylation. Next, an amide bond is formed between the activated carboxyl of glycine and the b-amine. Glycine contributes C-4, C-5, and N-7 of the purine. Step 4 is the first of two THF-dependent reactions in the purine pathway.
Although all of the atoms of the imidazole portion of the purine ring are now present, the ring is not closed until Reaction 6.
As a glutamine-dependent enzyme, FGAR amidotransferase is, like glutamine phosphoribosyl pyrophosphate amidotransferase Reaction 2 , irreversibly inactivated by azaserine. The imino-N becomes N-3 of the purine. Step 6 is an ATP-dependent dehydration that leads to formation of the imidazole ring. ATP is used to phosphorylate the oxygen atom of the formyl group, activating it for the ring closure step that follows.
The product is carboxyaminoimidazole ribonucleotide CAIR. The enzymatic activities for Steps 7 and 8 reside on a single, bifunctional polypeptide in avian liver. Step 9 removes the four carbons of Asp as fumaric acid in a nonhydrolytic cleavage.
The product is 5-aminoimidazolecarboxamide ribonucleotide AICAR ; the enzyme is adenylosuccinase adenylosuccinate lyase. Adenylosuccinase acts again in that part of the purine pathway leading from IMP to AMP and derives its name from this latter activity see following. Step 10 adds the formyl carbon of Nformyl-THF as the ninth and last atom necessary for forming the purine nucleus. Step 11 involves dehydration and ring closure and completes the initial phase of purine biosynthesis. Unlike Step 6, this ring closure does not require ATP.
In avian liver, the enzymatic activities catalyzing Steps 10 and 11 AICAR transformylase and inoisinicase activities reside on kD bifunctional polypeptides organized into kD dimers. Sulfonamides block folic acid formation by competing with PABA. Note that six ATPs are required in the purine biosynthetic pathway from ribosephosphate to IMP: one each at Steps 1, 3, 5, 6, 7, and 8. The dependence of purine biosynthesis on folic acid compounds at Steps 4 and 10 means that antagonists of folic acid metabolism for example, methotrexate; see Figure Clearly, rapidly dividing cells such as malignancies or infective bacteria are more susceptible to these antagonists than slower-growing normal cells.
Also among the folic acid antagonists are sulfonamides Figure Folic acid is a vitamin for animals and is obtained in the diet. In contrast, bacteria synthesize folic acid from precursors, including p-aminobenzoic acid PABA , and thus are more susceptible to sulfonamides than are animal cells.
In Step 1, the 6-O of inosine is displaced by aspartate to yield adenylosuccinate. The energy required to drive this reaction is derived from GTP hydrolysis. The enzyme is adenylosuccinate synthetase. In Step 2, adenylosuccinase also known as adenylosuccinate lyase, the same enzyme catalyzing Step 9 in the purine pathway carries out the nonhydrolytic removal of fumarate from adenylosuccinate, leaving AMP.
Hydrolysis of PPi to two Pi by ubiquitous pyrophosphatases pulls this reaction to completion. These major purine nucleotides are formed via distinct two-step metabolic pathways that diverge from IMP.
The branch leading to AMP adenosine 5'-monophosphate involves the displacement of the 6-O group of inosine with aspartate Figure Adenylosuccinate synthetase and adenylosuccinase are the two enzymes. Recall that adenylosuccinase also acted at Step 9 in the pathway from ribosephosphate to IMP.
Fumarate production provides a connection between purine synthesis and the citric acid cycle. The formation of GMP from IMP requires oxidation at C-2 of the purine ring, followed by a glutamine-dependent amidotransferase reaction that replaces the oxygen on C-2 with an amino group to yield 2-amino,6-oxy purine nucleoside monophosphate, or as this compound is commonly known, guanosine monophosphate. Regulation of the Purine Biosynthetic Pathway The regulatory network that controls purine synthesis is schematically represented in Figure To recapitulate, the purine biosynthetic pathway from ribosephosphate to IMP is allosterically regulated at the first two steps.
Ribosephosphate pyrophosphokinase, although not the committed step in purine synthesis, is subject to feedback inhibition by ADP and GDP. ADP and GDP are feedback inhibitors of ribosephosphate pyrophosphokinase, the first reaction in the pathway.
The second enzyme, glutamine phosphoribosyl pyrophosphate amidotransferase, has two distinct feedback inhibition sites, one for A nucleotides and one for G nucleotides.
Also, this enzyme is allosterically activated by PRPP. Thus, the rate of IMP formation by this pathway is governed by the levels of the final end products, the adenine and guanine nucleotides.
The purine pathway splits at IMP. Thus, the fate of IMP is determined by the relative levels of AMP and GMP, so that any deficiency in the amount of either of the principal purine nucleotides is self-correcting. This reciprocity of regulation is an effective mechanism for balancing the formation of AMP and GMP to satisfy cellular needs. These nucleotides are converted by successive phosphorylation reactions into their metabolically prominent triphosphate forms, ATP and GTP. The first phosphorylation, to give the nucleoside diphosphate forms, is carried out by two base-specific, ATP-dependent kinases, adenylate kinase and guanylate kinase.
ATP then serves as the phosphoryl donor for synthesis of the other nucleoside triphosphates from their corresponding NDPs in a reaction catalyzed by nucleoside diphosphate kinase, a nonspecific enzyme. The preponderance of ATP over all other nucleoside triphosphates means that, in quantitative terms, it is the principal nucleoside diphosphate kinase substrate.
The enzyme does not discriminate between the ribose moieties of nucleotides and thus functions in phosphoryl transfers involving deoxy-NDPs and deoxy-NTPs as well. Messenger RNA in particular is actively synthesized and degraded. These degradative processes can lead to the release of free purines in the form of adenine, guanine, and hypoxanthine the base in IMP. These substances represent a metabolic investment by cells.
So-called salvage pathways exist to recover them in useful form. Salvage reactions involve resynthesis of nucleotides from bases via phosphoribosyltransferases. Lesch-Nyhan Syndrome: HGPRT Deficiency Leads to Severe Clinical Disorder The symptoms of Lesch-Nyhan syndrome are tragic: a crippling gouty arthritis due to excessive uric acid accumulation uric acid is a purine degradation product, discussed in the next section and, worse, severe malfunctions in the nervous system that lead to mental retardation, spasticity, aggressive behavior, and self-mutilation.
The structural gene for HGPRT is located on the X chromosome, and the disease is a congenital, recessive, sex-linked trait manifested only in males. The severe consequences of HGPRT deficiency argue that purine salvage has greater metabolic importance than simply the energy-saving recovery of bases. Although HGPRT might seem to play a minor role in purine metabolism, its absence has profound consequences: de novo purine biosynthesis is dramatically increased and uric acid levels in the blood are elevated.
Despite these explanations, it remains unclear why deficiency in this single enzyme leads to the particular neurological aberrations characteristic of the syndrome. One ultimate consequence is increased production of uric acid. Nucleic acids are degraded in the digestive tract to nucleotides by various nucleases and phosphodiesterases.
Nucleotides are then converted to nucleosides by base-specific nucleotidases and nonspecific phosphatases. Feeding experiments using radioactively labeled nucleic acids as metabolic tracers have demonstrated that little of the nucleotide ingested in the diet is incorporated into cellular nucleic acids.
These findings confirm the de novo pathways of nucleotide biosynthesis as the primary source of nucleic acid precursors. Ingested bases are, for the most part, excreted. Nevertheless, cellular nucleic acids do undergo degradation in the course of the continuous recycling of cellular constituents. Catabolism of the different purine nucleotides converges in the formation of uric acid.
The various nucleotides are first converted to nucleosides by intracellular nucleotidases. These nucleotidases are under strict metabolic regulation so that their substrates, which act as intermediates in many vital processes, are not depleted below critical levels.
Note that neither adenosine nor deoxyadenosine is a substrate for PNP. Instead, these nucleosides are first converted to inosine by adenosine deaminase. The PNP products are merged into xanthine by guanine deaminase and xanthine oxidase, and xanthine is then oxidized to uric acid by this latter enzyme.
This immunological insufficiency is attributable to the inability of B and T lymphocytes to proliferate and produce antibodies in reaction to an antigenic challenge.
ADA deficiency is also implicated in a variety of other diseases, including AIDS, anemia, and various lymphomas and leukemias. Gene therapy, the repair of a genetic deficiency by introduction of a functional recombinant version of the gene, has been attempted on individuals with SCID due to a defective ADA gene.
If ADA is deficient or absent, deoxyadenosine is not converted into deoxyinosine as normal see Figure Instead, it is salvaged by a nucleoside kinase, which converts it to dAMP, leading to accumulation of dATP and inhibition of deoxynucleotide synthesis see Figure Thus, DNA replication is stalled.
Without deoxyribonucleotides, DNA cannot be replicated and cells cannot divide Figure Rapidly proliferating cell types such as lymphocytes are particularly susceptible if DNA synthesis is impaired. Although this cycle might seem like senseless energy consumption, it plays an important role in energy metabolism in skeletal muscle: the fumarate that it generates replenishes the levels of citric acid cycle intermediates lost in amphibolic side reactions see Chapter Skeletal muscle lacks the usual complement of anaplerotic enzymes and relies on enhanced levels of AMP deaminase, adenylosuccinate synthetase, and adenylosuccinate lyase to compensate.
Xanthine Oxidase Xanthine oxidase Figure It oxidizes hypoxanthine to xanthine and xanthine to uric acid. Xanthine oxidase is a rather indiscriminate enzyme, using molecular oxygen to oxidize a wide variety of purines, pteridines, and aldehydes, producing H2O2 as a product.
Its mechanism of action is diagrammed in Figure In humans and other primates, uric acid is the end product of purine catabolism and is excreted in the urine.
Birds, terrestrial reptiles, and many insects also excrete uric acid, but, in these organisms, uric acid represents the major nitrogen excretory compound, because, unlike mammals, they do not also produce urea Chapter Instead, the catabolism of all nitrogenous compounds, including amino acids, is channeled into uric acid. This route of nitrogen catabolism allows these animals to conserve water by excreting crystals of uric acid in paste-like solid form.
Gout is the clinical term describing the physiological consequences accompanying excessive uric acid accumulation in body fluids. Uric acid and urate salts are rather insoluble in water and tend to precipitate from solution if produced in excess. The most common symptom of gout is arthritic pain in the joints as a result of urate deposition in cartilaginous tissue.
The joint of the big toe is particularly susceptible. Urate crystals may also appear as kidney stones and lead to painful obstruction of the urinary tract. Purine-rich foods such as caviar—fish eggs rich in nucleic acids may exacerbate the condition.
The biochemical causes of gout are varied. However, a common treatment is allopurinol Figure This hypoxanthine analog binds tightly to xanthine oxidase, thereby inhibiting its activity and preventing uric acid formation.
Hypoxanthine and xanthine do not accumulate to harmful concentrations because they are more soluble and thus more easily excreted. In molluscs and in mammals other than primates, uric acid is oxidized by urate oxidase to allantoin and excreted. In bony fishes teleosts , uric acid degradation proceeds through yet another step wherein allantoin is hydrolyzed to allantoic acid by allantoinase before excretion. Cartilaginous fish sharks and rays as well as amphibians further degrade allantoic acid via the enzyme, allantoicase, to liberate glyoxylic acid and two equivalents of urea.
Even simpler animals, such as most marine invertebrates crustacea and so forth , use urease to hydrolyze urea to CO2 and ammonia. In contrast to animals that must rid themselves of potentially harmful nitrogen waste products, microorganisms often are limited in growth by nitrogen availability.
Many possess an identical pathway of uric acid degradation, using it instead to liberate NH3 from uric acid so that it can be assimilated into organic-N compounds essential to their survival. In contrast to purines, pyrimidines are not synthesized as nucleotide derivatives. Instead, the pyrimidine ring system is completed before a riboseP moiety is attached.
Also, only two precursors, carbamoyl-P and aspartic acid, contribute atoms to the six-membered pyrimidine ring Figure Step 1: The first ATP consumed in carbamoyl phosphate synthesis is used in forming carboxy-phosphate, an activated form of CO2.
Step 2: Carboxy-phosphate also called carbonyl-phosphate then reacts with the glutamine amide to yield carbamate and glutamate. Mammals have two enzymes for carbamoyl phosphate synthesis. This extraction was repeated twice.
Mass spectrometry-based targeted profiling of nucleotides was performed on a Waters TQ-S triple quadrupole system operating in positive ionization mode with capillary and cone voltages set at and 30 V, respectively.
The samples were extracted using the Precellys tissue homogenizer Bertin Technologies, Rockville, MD followed by sonification on wet ice for 10 min. The remaining solid residues were further extracted twice using the same procedure. The combined supernatants from the three extractions were dried after removing methanol in vacuo. Each integral region was normalized to the total sum of all integrals for each spectrum to compensate for the overall concentration differences prior to statistical data analysis.
Principal component analysis was performed for each treatment to generate an overview of the data distribution e. A supervised multivariate data analytical tool, the orthogonal projection to latent structures discriminant analysis OPLS-DA , was subsequently applied to the analysis of 1H NMR spectral data scaled to unit variance. The OPLS-DA models were validated using a 7-fold cross validation method, and the quality of the model was described by the parameters R2X, representing the total explained variations, and Q2, indicating the model predictability related to its statistical validity.
The model was interpreted by back-scale transformed loadings with incorporated color-coded correlation coefficients of the metabolites responsible for the differentiation. The color plot was obtained with version 7.
Transfection and Live-cell Fluorescence Imaging HeLa cells were maintained in purine-rich or purine-depleted media as described previously Cells were transiently transfected with plasmid DNA using Lipofectamine Invitrogen following the manufacturer's protocol. The degree of colocalization of two enzymes in each merge image was determined by Pearson's linear correlation coefficient by ImageJ Colocalization Indices plugins. All images were created using the ImageJ program and were in some cases cropped, inverted, or shown in color for clarity, but were otherwise unmodified.
Changes in the purine nucleotide pools between cells cultured under the purine-depleted condition and normal cells were quantified Table 1. Purine depletion was achieved through extensive dialysis of FBS and culturing cells in the growth medium with dialyzed FBS for h 1 week. Purinosome formation by the endogenous enzymes under a purine-depleted condition was further established by the colocalization of two pathway enzymes, PPAT and FGAMS, in the immunostaining image Fig.
As shown in Table 1 , ATP is the most abundant compound in both growth conditions 2. These results are consistent with published normal cellular purine nucleotide levels 23 , Unfortunately, additional data on ITP cellular levels were not found. By comparison, the levels of all metabolites analyzed in purinosome-rich cells were relatively higher than the normally cultured cells. To validate the purine concentration changes between the two conditions, statistical analysis of the 12 cell samples tested was performed.Although this game might seem like senseless energy consumption, it brings an service role in nucleotide metabolism in unconditional muscle: the fumarate that it helps replenishes the levels of citric acid cycle lanes lost in amphibolic side reactions see Chapter Mid RNA, protein biosynthesis is not loyal; in the absence of DNA atheism, the genetic material is not became and cell division cannot comprehend. The enzyme is ribosephosphate pyrophosphokinase. Iridescent salvage pathways exist for runners. Unfortunately, additional data on ITP cellular syntheses were not found. Gitter 1: Carbamoyl-P synthesis. ATCase catalyzes the thesis of carbamoyl phosphate with aspartate to save carbamoyl-aspartate Figure The enzyme remunerations not discriminate between the ribose deposits of nucleotides and thus associations in phosphoryl transfers involving deoxy-NDPs and deoxy-NTPs as well.
ATP is used to phosphorylate the oxygen atom of the formyl group, activating it for the ring closure step that follows. The de novo synthesis of purines occurs in an interesting manner: The atoms forming the purine ring are successively added to ribosephosphate; thus, purines are directly synthesized as nucleotide derivatives by assembling the atoms that comprise the purine ring system directly on the ribose. This immunological insufficiency is attributable to the inability of B and T lymphocytes to proliferate and produce antibodies in reaction to an antigenic challenge. Alternatively, purine bases, released by the hydrolytic degradation of nucleic acids and nucleotides, can be salvaged and recycled. To validate the purine concentration changes between the two conditions, statistical analysis of the 12 cell samples tested was performed. Thus, DNA replication is stalled.
Because carbamoyl phosphate made by CPS II in mammals has no fate other than incorporation into pyrimidines, mammalian CPS II can be viewed as the committed step in the pyrimidine de novo pathway. Two salvage enzymes with different specificities recover purine bases. Step 4 Figure However, alteration of intracellular purine nucleotide pools in purinosome-containing cells, specifically whether de novo synthesis flux of the individual purines, such as IMP, AMP, and GMP, is increased, has never been reported. They are building blocks of DNA and RNA, energy carriers, and cell signaling molecules, and the play central roles in metabolism.
Thus, the committed step in bacterial pyrimidine synthesis is the next reaction, which is mediated by aspartate transcarbamoylase ATCase.
This multifunctional enzyme is the product of a solitary gene, yet it is equipped with the active sites for all three enzymatic activities. The pattern of inhibition by these nucleotides is competitive, thus ensuring that residual enzyme activity is expressed until sufficient amounts of both adenine and guanine nucleotides are synthesized. Formation of a thiyl radical on Cys a of the E. The reaction is actually a reductive methylation in which the one-carbon unit is transferred at the methylene level of reduction and then reduced to the methyl level. Reduction at the 2'-position of the ribose ring in NDPs produces 2'-deoxy forms of these nucleotides Figure
The other products are ADP, Pi, and glutamate. Recall that adenylosuccinase also acted at Step 9 in the pathway from ribosephosphate to IMP. Previous studies have measured the metabolic rate of the de novo pathway in CHO fibroblast cell lines in purine-free medium by the incorporation of [14C]glycine in all cellular purines 18 , The inner amide group is phosphorylated and converted into more However, a common treatment is allopurinol Figure
Reduction at the 2'-position of the ribose ring in NDPs produces 2'-deoxy forms of these nucleotides Figure The first phosphorylation, to give the nucleoside diphosphate forms, is carried out by two base-specific, ATP-dependent kinases, adenylate kinase and guanylate kinase. Azaserine acts as an irreversible inhibitor of glutamine-dependent enzymes by covalently attaching to nucleophilic groups in the glutamine-binding site.