Enzymes nucleotide biosynthesis

Inosine monophosphate dehydrogenase (EVDPDH) is a key enzyme of purine nucleotide biosynthesis. Purine synthesis in lymphocytes exclusively depends on the de novo synthesis, whereas other cells can generate purines via the so-called salvage pathway. Therefore, IMPDH inhibitors preferentially suppress DNA synthesis in activated lymphocytes. 

Inosine monophosphate dehydrogenase (IMPDH) is the key enzyme of purine nucleotide biosynthesis. Proliferation of activated lymphocytes dq ends on rapid de novo production of purine nucleotides for DNA synthesis. 

Synthetic analogs of purine and pyrimidine bases and their derivatives serve as anticancer dmgs either by inhibiting an enzyme of nucleotide biosynthesis or by being incorporated into DNA or RNA. 

Since biosynthesis of IMP consumes glycine, glutamine, tetrahydrofolate derivatives, aspartate, and ATP, it is advantageous to regulate purine biosynthesis. The major determinant of the rate of de novo purine nucleotide biosynthesis is the concentration of PRPP, whose pool size depends on its rates of synthesis, utilization, and degradation. The rate of PRPP synthesis depends on the availabihty of ribose 5-phosphate and on the activity of PRPP synthase, an enzyme sensitive to feedback inhibition by AMP, ADP, GMP, and GDP. 

Figure 34-7 summarizes the roles of the intermediates and enzymes of pyrimidine nucleotide biosynthesis. The catalyst for the initial reaction is cytosolic carbamoyl phosphate synthase II, a different enzyme from the mitochondrial carbamoyl phosphate synthase I of urea synthesis (Figure 29-9). Compartmentation thus provides two independent pools of carbamoyl phosphate. PRPP, an early participant in purine nucleotide synthesis (Figure 34-2), is a much later participant in pyrimidine biosynthesis.

Certain Pyrimidine Analogs Are Substrates for Enzymes of Pyrimidine Nucleotide Biosynthesis... 

The activities of the first and second enzymes of pyrimidine nucleotide biosynthesis are controlled by allosteric... 

The first step of this sequence, which is not unique to de novo purine nucleotide biosynthesis, is the synthesis of 5-phosphoribosylpyrophosphate (PRPP) from ribose-5-phosphate and adenosine triphosphate. Phosphoribosyl-pyrophosphate synthetase, the enzyme that catalyses this reaction [278], is under feedback control by adenosine triphosphate [279]. Cordycepin interferes with thede novo pathway [229, 280, 281), and cordycepin triphosphate inhibits the synthesis of PRPP in extracts from Ehrlich ascites tumour cells [282]. Formycin [283], probably as the triphosphate, 9-0-D-xylofuranosyladenine [157] triphosphate, and decoyinine (LXXlll) [284-286] (p. 89) also inhibit the synthesis of PRPP in tumour cells, and this is held to be the blockade most important to their cytotoxic action. It has been suggested but not established that tubercidin (triphosphate) may also be an inhibitor of this reaction [193]. 

Glutamine also supplies an amino function to start off purine nucleotide biosynthesis. This complex little reaction is again an Sn2 reaction on PRPP, but only an amino group from the amide of glutamine is transferred. The product of the enzymic reaction is thus 5-phosphoribosylamine. 

The nucleotides are among the most complex metabolites. Nucleotide biosynthesis is elaborate and requires a high energy input (see p. 188). Understandably, therefore, bases and nucleotides are not completely degraded, but instead mostly recycled. This is particularly true of the purine bases adenine and guanine. In the animal organism, some 90% of these bases are converted back into nucleoside monophosphates by linkage with phosphori-bosyl diphosphate (PRPP) (enzymes [1] and [2]). The proportion of pyrimidine bases that are recycled is much smaller. 

Antimetabolites are enzyme inhibitors (see p. 96) that selectively block metabolic pathways. The majority of clinically important cytostatic drugs act on nucleotide biosynthesis. Many of these are modified nucleobases or nucleotides that competitively inhibit their target enzymes (see p. 96). Many are also incorporated into the DNA, thereby preventing replication. 

As the first committed step in the biosynthesis of AMP from IMP, AMPSase plays a central role in de novo purine nucleotide biosynthesis. A 6-phosphoryl-IMP intermediate appears to be formed during catalysis, and kinetic studies of E. coli AMPSase demonstrated that the substrates bind to the enzyme active sites randomly. With mammalian AMPSase, aspartate exhibits preferred binding to the E GTPTMP complex rather than to the free enzyme. Other kinetic data support the inference that Mg-aspartate complex formation occurs within the adenylosuccinate synthetase active site and that such a... 

TThe growth of cancer cells is not controlled in the same way as cell growth in most normal tissues. Cancer cells have greater requirements for nucleotides as precursors of DNA and RNA, and consequently are generally more sensitive than normal cells to inhibitors of nucleotide biosynthesis. A growing array of important chemotherapeutic agents—for cancer and other diseases—act by inhibiting one or more enzymes in these pathways. We describe here several well-studied examples that illustrate productive approaches to treatment and help us understand how these enzymes work. 

Synthesis of 5 phosphoribosylamine from PRPP and glutamine is catalized by glutamine phosphoribosyl pyrophosphate amidotransferase. This enzyme is inhibited by the purine 5 -nucleotides, AMP, GMP, and IMP—the end-products of the pathway. This is the committed step in purine nucleotide biosynthesis. 

Acivicin is a potent inhibitor of several steps in purine nucleotide biosynthesis that utilize glutamine. The enzymes it inhibits are glutamine PRPP amidotransferase (step 1, fig. 23.10), phosphoribosyl-A-formylglycinamidine synthase (step 4, fig. 23.10), and GMP synthase (see fig. 23.11). In pyrimidine nucleotide biosynthesis the enzymes inhibited are carbamoyl synthase (step 1, fig. 23.13) and CTP synthase (see fig. 23.14). Acivicin is under trial for the treatment of some forms of cancer. 

Another group of inhibitors prevents nucleotide biosynthesis indirectly by depleting the level of intracellular tetrahydrofolate derivatives. Sulfonamides are structural analogs of p-aminobenzoic acid (fig. 23.19), and they competitively inhibit the bacterial biosynthesis of folic acid at a step in which p-aminobenzoic acid is incorporated into folic acid. Sulfonamides are widely used in medicine because they inhibit growth of many bacteria. When cultures of susceptible bacteria are treated with sulfonamides, they accumulate 4-carboxamide-5-aminoimidazole in the medium, because of a lack of 10-formyltetrahydrofolate for the penultimate step in the pathway to IMP (see fig. 23.10). Methotrexate, and a number of related compounds inhibit the reduction of dihydrofolate to tetrahydrofolate, a reaction catalyzed by dihydrofolate reductase. These inhibitors are structural analogs of folic acid (see fig. 23.19) and bind at the catalytic site of dihydrofolate reductase, an enzyme catalyzing one of the steps in the cycle of reactions involved in thymidylate synthesis (see fig. 23.16). These inhibitors therefore prevent synthesis of thymidylate in replicating... 

Many lines of evidence indicate that the first committed step in de novo purine nucleotide biosynthesis, production of 5-phosphoribosylamine by glutamine PRPP amidotransfer-ase, is rate-limiting for the entire sequence. Consequently, regulation of this enzyme is probably the most important factor in control of purine synthesis de novo (fig. 23.24). The enzyme is inhibited by purine-5 -nucleotides, but the most inhibitory nucleotides vary with the source of the enzyme. Inhibition constants (A, ) are usually in the range 10-3-10-5 M. The maximum effect of this end-product inhibition is produced by certain combinations of nucleotides (e.g., AMP and GMP) in optimum concentrations and ratios, indicating two kinds of inhibitor binding sites. This is an example of a concerted feedback inhibition. 

Jones, M. E., Pyrimidine nucleotide biosynthesis in animals Genes, enzymes and regulation of UMP biosynthesis. Ann. Rev. Biochem. 49 253-279, 1980. Authoritative outline of the regulatory properties of the two multifunctional proteins responsible for pyrimidine nucleotide synthesis in animals. 

Fe/S clusters in regulatory enzymes have been proposed to act as sensors in such a manner that, upon detection of a measurand, the cluster disintegrates and activity stops. Putative examples are NO sensing by the [2Fe-2S] cluster in the terminal enzyme of heme synthesis, ferrochelatase [8], and 02 sensing by the [4Fe-4S] cluster in the regulatory enzyme of purine nucleotide biosynthesis, glutamine 5-phosphoribosyl-l-pyrophosphate amidotransferase [9], This is of course not a catalytic activity, since the cluster is destroyed in the action. 

B lymphocytes will be eliminated during continuous culture because these cells have a short life span in culture. Commercially available myeloma cells for hybridoma production have mutations in one of the enzymes of the salvage pathway of purine nucleotide biosynthesis. Hybridoma cells are cultured in medium that forces the cells to utilize the salvage pathway for nucleotide synthesis. The mutated myeloma cells or hybridization products of two myeloma cells will die in this selection medium since they are incapable of nucleotide synthesis under these propagation conditions. However, myeloma cells that have fused to the B lymphocytes derived from the spleen of the immunized animal will have an intact salvage pathway and will survive in the selection medium. Thus, only the B lymphocytes-myeloma hybridomas will survive prolonged culture in the selection medium. 

The enzymes that catalyze these steps are homologous to argininosuccinate synthetase and argininosuccinase, respectively. Thus, four of the five enzymes in the urea cycle were adapted from enzymes taking part in nucleotide biosynthesis. The remaining enzyme, arginase, appears to be an ancient enzyme found in all domains of life. 

Nucleotide biosynthesis is regulated by feedback inhibition in a manner similar to the regulation of amino acid biosynthesis (Section 24,3). Indeed, aspartate transcarbamoylase, one of the key enzymes for the regulation of pyrimidine biosynthesis in bacteria, was described in detail in Chapter 10. Recall ihaiATCase is inhibited by CTP, the final product ofpyrimidine biosynthesis, and stimulated by ATP. Carbamoyl phosphate synthetase is a site of feedback inhibition in both prokaryotes and eukaryotes. 

The committed step in purine nucleotide biosynthesis is the conversion of PRPP into phosphoribosylamine by glutamine phosphoribosyl amidotransferase. This important enzyme is feedback-inhibited by many purine ribonucleotides. It is noteworthy that AMP and GMP, the final products of the pathway, are synergistic in inhibiting the amidotransferase. 

V.M. Levdikov, V.V. Barynin, Al. Grebenko, W.R. Melik-Adamyan, V.S. Lamzin, and K.S. Wilson. 1998. The structure of SAICAR synthase An enzyme in the de novo pathway of purine nucleotide biosynthesis Structure 6 363-376. (PubMed)... 

In eukaryotic cells, two separate pools of carbamoyl phosphate are synthesized by different enzymes located at different sites. Carbamoyl phosphate synthetase I (CPS I) is located in the inner membrane of mitochondria in the liver and, to lesser extent, in the kidneys and small intestine. It supplies carbamoyl phosphate for the urea cycle. CPS 1 is specific for ammonia as nitrogen donor and requires N-acetylglutamate as activator. Carbamoyl phosphate synthetase II (CPS II) is present in the cytosol. It supplies carbamoyl phosphate for pyrimidine nucleotide biosynthesis and uses the amido group of glutamine as nitrogen donor. The presence of physically separated CPSs in eukaryotes probably reflects the need for independent regulation of pyrimidine biosynthesis and urea formation, despite the fact that both pathways require carbamoyl phosphate. In prokaryotes, one CPS serves both pathways. 

PRPP affects purine and pyrimidine nucleotide biosynthesis. PRPP formation is activated by inorganic phosphate and inhibited by several end products of pathways that use PRPP. In the purine de novo pathway, PRPP activates amidophosphoribosyltransferase and is the rate-limiting substrate for the enzyme. In purine... 

The answer is c. (Murray, pp 375-401. Scriver, pp 2513-2570. Sack, pp 121-138. Wilson, pp 287-320.) The steps of pyrimicfine nucleotide biosynthesis are summarized in the figure below. The first step in pyrimidine synthesis is the formation of carbamoyl phosphate. The enzyme catalyzing this step, carbamoyl phosphate synthetase (1), is feedback-inhibited by UMP through allosteric effects on enzyme structure (not by competitive inhibition with its substrates). The enzyme of the second step, aspartate transcarbamoylase, is composed of catalytic and regulatory subunits. The regulatory subunit binds CTP or ATP TTP has no role in the feedback inhibition of pyrimidine synthesis. Decreased rather than increased activity of enzymes 1 and 2 would be produced by allosteric feedback inhibition. 

Despite their absence from the current sequence classification, much is known about the mechanisms of A -glycoside formation, hydrolysis and phos-phorolysis at the anomeric centre of o-ribofuranose. The enzymes physiologically are involved in nucleoside and nucleotide biosynthesis and degradation some work at the nucleic acid polymer level to remove or add modified nucleic acid bases. 

Tetrahydrofolate (Fig. le) and derivatives (Fig. If, g) are the biologically active forms of folate. This cofactor is involved in many, distinct enzymatic reactions, ranging from the amino acid metabolism, such as serine hydroxymethyltransferase (SHMT) (Fig. 5a), to nucleotide biosynthesis, such as thymidylate synthase (TS) (Fig. 5b) and dihydrofolate reductase DHFR (Fig. 5c). These enzymes are targets for anticancer drugs because they participate in the formation of thymidylate, the only nucleotide that cannot be obtained via the salvage reactions (30). Whereas the search for inhibitors of SHTM has only recently... 

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