IMP synthesis

The rate of the amidotransferase reaction is also governed by intracellular concentrations of the substrates l-glutamine and PRPP. Competing metabolic reactions or drugs that alter the supply of these substrates also affect the rate of IMP synthesis. 

Correcting deficiencies. Suppose that a person is found who is defrcient in an enzyme required for IMP synthesis. How might this person be treated ... 

In order to focus on the IMP synthesis reaction described above, we could lay out the subgraph surrounding the reaction node ... 

Other factors, however, may also be important. Edwards and co workers (15) have shown that the decreased reutilization of hypoanthine in patients with HPRT deficiency results in the loss of a normal major source of intracellular nucleotide. This drain of hypo-xanthine from the cell tends to decrease the consumption of PRPP and reduce the intracellular concentration of IMP. The elevated concentration of PRPP in turn presumably allows the cell to compensate for the loss of IMP by virtue of the accelerated rate of IMP synthesis. 

GMP inhibited both adenine and guanine nucleotide synthesis (Table II compare assays (ii) and (v) (iii) and (vi) (vii) and (ix)). The branch point regulation i.e. inhibition of adenylosuccinate synthetase (EC 6.3.4.4) and IMP dehydrogenase (EC 1.2.1.13) may be more apparent than real due to the extremely low rate of IMP synthesis secondary to proximal pathway inhibition. GTP apparently also inhibited IMP dehydrogenase as the synthesis of guanine nucleotides was significantly decreased in the assay for simultaneous adenine and guanine nucleotide synthesis (Table II compare assays (iii) and (vii)). 

Tissues were homogenized in phosphate buffer and sonicated, and the supernatant was used for electrophoresis. Samples were run in phosphate buffer, pH 8.5, on cellulose acetate paper for 2 hours at 4 and at 0.5 mA/cm. PRPP synthetase was located on the paper by a radiochemical assay formation of PRPP from ribose-5-phosphate and ATP was coupled to inosinic acid (IMP) synthesis by the addition to the reaction mixture of labelled hypoxanthine and partially purified hypoxan-thine-guanine phosphoribosyltransferase (HGPRT). 

P5C stimulated IMP synthesis from hypoxanthine determined not only by an increased incorporation of labeled hypoxanthine but also by increased total IMP pools in red cells (Fig. 8). When treated with P5C, red cells incubated with hypoxanthine attained levels of IMP which were almost 30% of the total purine nucleotide complement (118), levels which are 10-fold those found in circulating red cells. Whether the augmentation of nucleotide pools physiologically affects red cells, themselves, or functions mediated by red cells, e.g., the transfer of purines between tissues remains to be ascertained. 

Bredinin, Neosidomycin, and SF-2140. Bredinin (62), isolated from the culture filtrates of Eupenicillium brefeldianum (1,4), inhibits the multiplication of L5178Y, HeLa S3, RK-13, mouse L-ceUs, and Chinese hamster cells. GMP can reverse the inhibition by (62), but (62) is not incorporated into the nucleic acids. The inhibition of nucleic acid synthesis and chromosomal damage in the S and G 2 phases that is caused by (62), is reversed by GMP. It blocks the conversion of IMP to XMP and XMP to GMP. In combination with GMP, (62) interferes with intracellular cAMP levels and thereby inhibits cell division. 

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. 

The free bases of the purines can be salvaged to spare de novo synthesis. The only hard thing is remembering what the names stand for. HGPRTase is hypoxanthine-guanine phosphoribosyltransferase, and it makes both IMP and GMP. A separate enzyme exists for the salvage of adenine. The salvage pathways are included in Fig. 19-1. 

Within a cell, a nncleotidase catalyses the hydrolysis of either a ribonncleotide or deoxyribonucleotide (Fignre 10.8). The qnantitatively important pathway for degradation of AMP in liver and mnscle involves deamination to IMP, catalysed by AMP deaminase, producing ammonia, and snbseqnent hydrolysis of IMP to inosine. This may be an important sonrce of inosine for synthesis of phosphati-dylinositol, a key phospholipid in membranes. 

Figure 20.8 Summary of pathways for de novo synthesis of purine and pyrimidine nucleotides. C represents transfer of a single carbon atom (a one-carbon transfer). Details are provided in Appendix 20.1. IMP - inosine monophosphate. For thymi-dylate synthesis, see Figure 20.12a.

In the purine nucleotide pathway, the purine nucleotide is synthesised upon the phosphoribose using several small molecules. The first purine nucleotide formed is inosine monophosphate (IMP) it is an intermediate on the pathway for the synthesis of both adenine and guanine nucleotides (Figure 20.8). 

The major intermediates in the biosynthesis of nucleic acid components are the mononucleotides uridine monophosphate (UMP) in the pyrimidine series and inosine monophosphate (IMP, base hypoxanthine) in the purines. The synthetic pathways for pyrimidines and purines are fundamentally different. For the pyrimidines, the pyrimidine ring is first constructed and then linked to ribose 5 -phosphate to form a nucleotide. By contrast, synthesis of the purines starts directly from ribose 5 -phosphate. The ring is then built up step by step on this carrier molecule. 

De novo synthesis of purines and pyrimidines yields the monophosphates IMP and UMP, respectively (see p. 188). All other nucleotides and deoxynucleotides are synthesized from these two precursors. An overview of the pathways involved is presented here further details are given on p. 417. Nucleotide synthesis by recycling of bases (the salvage pathway) is discussed on p. 186. 

The synthesis of purine nucleotides (1) starts from IMP. The base it contains, hypoxanthine, is converted in two steps each into adenine or guanine. The nucleoside monophosphates AMP and CMP that are formed are then phos-phorylated by nucleoside phosphate kinases to yield the diphosphates ADP and GDP, and these are finally phosphorylated into the triphosphates ATP and CTP. The nucleoside triphosphates serve as components for RNA, or function as coenzymes (see p. 106). Conversion of the ribonucleotides into deoxyribo-nucleotides occurs at the level of the diphosphates and is catalyzed by nucleoside diphosphate reductase (B). 

Muscle-specific auxiliary reactions for ATP synthesis exist in order to provide additional ATP in case of emergency. Creatine phosphate (see B) acts as a buffer for the ATP level. Another ATP-supplying reaction is catalyzed by adenylate kinase [1] (see also p.72). This disproportionates two molecules of ADP into ATP and AMP. The AMP is deaminated into IMP in a subsequent reaction [2] in order to shift the balance of the reversible reaction [1 ] in the direction of ATP formation. 

Figure 10-1. Overview of purine synthesis. Details of the first two reactions and sources of the atoms of the purine ring in inosine 5 -monophosphate (IMP) are shown. PRPP, 5 -phosphoribosyl-1-pyrophosphate Gin, glutamine Gly, glycine Asp, aspartate THF, tetrahydrofolate.

Figure 10-2. Regulation of purine synthesis by the nucleotides and the intermediate, 5 -phosphoribosyl-1 -pyrophosphate (PRPP). Both feedback and feed-forward mechanisms are utilized in this intricate scheme. IMP, inosine monophosphate.

Three major feedback mechanisms cooperate in regulating the overall rate of de novo purine nucleotide synthesis and the relative rates of formation of the two end products, adenylate and guanylate (Fig. 22-35). The first mechanism is exerted on the first reaction that is unique to purine synthesis—transfer of an amino group to PRPP to form 5-phosphoribosylamine. This reaction is catalyzed by the allosteric enzyme glutamine-PRPP amidotransferase, which is inhibited by the end products IMP, AMP, and GMP. AMP and GMP act synergisti-cally in this concerted inhibition. Thus, whenever either AMP or GMP accumulates to excess, the first step in its biosynthesis from PRPP is partially inhibited. 

In the second control mechanism, exerted at a later stage, an excess of GMP in the cell inhibits formation of xanthylate from inosinate by IMP dehydrogenase, without affecting the formation of AMP (Fig. 22-35). Conversely, an accumulation of adenylate inhibits formation of adenylosuccinate by adenylosuccinate synthetase, without affecting the biosynthesis of GMP. In the third mechanism, GTP is required in the conversion of IMP to AMP (Fig. 22-34, step (T)), whereas ATP is required for conversion of IMP to GMP (step (4)), a reciprocal arrangement that tends to balance the synthesis of the two ribonucleotides. 

The next nine steps in purine nucleotide biosynthesis leading to the synthesis of IMP (whose base is hypoxanthine) are illustrated in Figure 22.7. This pathway requires four ATP molecules as an energy source. Two steps in the pathway require N10-formyltetrahydrofolate. 

The conversion of IMP to either AMP or GMP uses a two-step, energy-requiring pathway (Figure 22.8). Note that the synthesis of AMP requires GTP as an energy source, whereas the synthesis of GMP requires ATP. Also, the first reaction in each pathway is inhib-... 

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