The Conversion of IMP to AMP

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. 

Next, in steps 7 and 8, N-l of the purine ring is contributed by aspartate. Aspartate forms an amide with the 4-carboxyl group, and the succinocarboxamide so formed is then cleaved with release of fumarate. Energy for carboxamide formation is provided by ATP hydrolysis to ADP and phosphate. These reactions resemble the conversion of cit-rulline to arginine in the urea cycle (chapter 22) and the conversion of IMP to AMP (see fig. 23.11). 

The conversion of IMP to AMP and GMP is shown in Figure 10.8. Note that GTP is required for the biosynthesis of AMP and ATP for GMP biosynthesis, and high-ATP levels channel the conversion of IMP to GMP. In addition, IMP dehydrogenase is inhibited by GMP. We therefore have additional loci for controlling cellular concentrations of purine nucleotides by regulating the fate of IMP. The conversion of nucleoside monophosphates to di- and triphosphates is discussed later. 

Mercaptopurine (6-thioxo-l,6-dihydropurine) was one of the first purine derivatives to find application in the treatment of leukaemia, especially in children. The drug inhibits the biosynthesis of purine nucleosides, including the conversion of IMP to AMP (54M140905, B-66MI40900). The 5-imidazolyl derivative (384), azathiopurine or Imvran, has also been of value as a suppressor of the immune response. 

IMP is the precursor of both AMP and GMP. The conversion of IMP to AMP takes place in two stages (Figure 23.21). The first step is the reaction of aspartate with IMP to form adenylosuccinate. This reaction is catalyzed by adenylosuccinate synthetase and requires GTP, not ATP, as an enei source (using ATP would be counterproductive). The cleavage of fumarate from adenylosuccinate to produce AMP is catalyzed by adenylosuccinase. This enzyme also functions in the synthesis of the six-membered ring of IMP. 

This reaction is the first committed step in the conversion of IMP to AMP and represents a branch point in the synthesis of IMP, the end product of tie novo purine biosynthesis. Although it had been known for some time that nitrogen from dietary components is rapidly incorporated into cellular AMP, especially into the 6-amino group 1, 2), specific s3mthesis of AMP from IMP was not demonstrated until 1955. Abrams and Bentley (3) showed that the conversion occurred in rabbit bone marrow while Lieberman (4) demonstrated it in Escherichia coli. Carter and Cohen (5, 6) isolated and characterized adenylosuccinate from yeast extracts. Lieberman (4) demonstrated that adenylosuccinate could be formed reversibly from IMP and aspartate by a partially purified enzyme fraction fromE. coli. 

The level of aspartate has been shown to influence the activity of adenylosuccinate synthetase in vivo both in Ehrlich ascites cells (26) and cultured fibroblasts 95). Addition of exogenous aspartate to either system increased the conversion of IMP to AMP and reduced breakdown of IMP to inosine and hypoxanthine 26, 95). 

The conversion of IMP to AMP requires amination at C-6 of the purine system, and the nitrogen for this process is derived from aspartate (Asp, D) (adenylosuccinate synthase, EC 6.3.4.4). It appears that the driving force for the loss of water in this process that yields N -l, 2-dicarboxyl adenosine monophosphate (adenosine 5 -phosphate, AMP) is the conversion of guanosine triphosphate (GTP) to guano-sine diphosphate (GDP). Adenosine monophosphate (adenosine 5 -phosphate, AMP) is formed from the N -derivative by loss of fumarate (catalyzed by adenylosuccinate lyase, EC 4.3.2.2). 

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-... 

Conversion of IMP to AMP and GMP. In both cases two steps are required. Note that the formation of AMP requires GTP, and the formation of GMP requires ATP. This tends to balance the flow of the IMP down the two pathways. 

The conversion of IMP to either adenosine monophosphate (AMP or adenylate) or guanosine monophosphate (GMP or guanylate) requires two reactions (Figure 14.25). 

Subsequent phosphorylation reactions produce purine nucleoside diphosphates (ADP and GDP) and triphosphates (ATP and GTP). The purine nucleoside monophosphates, diphosphates, and triphosphates are all feedback inhibitors of the first stages of their own biosynthesis. Also, AMP, ADP, and ATP inhibit the conversion of IMP to adenine nucleotides, and GMP, GDP, and GTP inhibit the conversion of IMP to xanthylate and to guanine nucleotides (Figure 23.22). 

The conversion of IMP to GMP produces one NADH and uses the equivalent of 2 ATP because an ATP is converted to AMP. Because NADH gives rise to 2.5 ATP if it goes into the electron transport chain, we can say that the conversion results in a net production of ATP. 

Adenylosuccinate formed by adenylosuccinate synthetase is cleaved by adenylosuccinate lyase to form AMP. The reaction steps are illustrated in Fig. 1. Included in the sequence is the additional reaction catalyzed by AMP deaminase. These three enzymes have been suggested to function in a cyclic process termed the purine nucleotide cycle 7,8). The two-step conversion of IMP to AMP is very similar to both the conversion of citrulline to arginine, which involves formation of argininosuccinate as an intermediate, and formation of 5-amino-imidazole 4-carboxamide ribonucleotide from 5-aminoimidazole 4-carboxylate ribonucleotide as part of IMP biosynthesis. Adenylosuccinate lyase is a dual function enzyme catalyzing the cleavage of both adenylosuccinate and 5-aminoimidazole 4-N-succinocarboxamide ribonucleotide. 

Regulation in tumor cells appears to allow more efficient conversion of IMP to AMP. The level of the synthetase is increased from 1.6- to 3.7-fold in a number of tumors irrespective of growth rate (23). The kinetic properties of the acidic isozymes from Walker 10) and Novikoff 45) tumors indicate decreased inhibition by AMP. The Km for IMP is decreased for the Novikoff enzyme while the Km for aspartate is increased 45). These changes would favor AMP synthesis as compared to nonneoplastic cells. 

Figure 20.13 Summary of the reactions by which all four deoxy-ribonucleoside triphosphates can be synthesised from the nucleosides, adenosine and uridine. The reactions are summaries of the processes presented in Figures 20.8, 20.9 and 20.12. AMP is converted to IMP by a deaminase (Chapter 6). The conversion of UTP to CTP is catalysed by CTP synthetase.

The answer is c. (Ivlurray, pp 375— /O I. Scrivt i, pp 2513—2570. Sack, pp 121—138. Wilson, pp 287—320.1 Several control sites exist in the path of purine synthesis where feedback inhibition occurs, AMP, GMP, or IMP may inhibit the first step of the pathway, which is the synthesis ol 5-phosphoribosyl-l-pyrophosphate (PRPP). PRPP synthetase is specifically inhibited. All three nucleotides can inhibit glutamine PRPP aminotranslerase, which catalyzes the second step of the. pathway. AMP blocks the conversion ol IMP to adenylosuccinate. GMP inhibits the lormation ol xanthylate Irom IMP Thus, blockage rather than enhancement ol IMP metabolism to AMP and GMP effectively inhibits purine biosynthesis. 

Figure 34-2 illustrates the intermediates and reactions for conversion of a-D-ribose 5-phosphate to inosine monophosphate (IMP). Separate branches then lead to AMP and GMP (Figure 34-3). Subsequent phosphoryl transfer from ATP converts AMP and GMP to ADP and GDP. Conversion of GDP to GTP involves a second phosphoryl transfer from ATP, whereas conversion of ADP to ATP is achieved primarily by oxidative phosphorylation (see Chapter 12).

While the formation of AMP from hypoxanthine is surely a good start, the salvage will be truly successful only if the AMP is converted to ATP. However, prior to continued phosphorylation, the AMP formed by the two-enzyme reaction sequence described above can undergo another fate—deamination to form IMP and ammonia (see Fig. 10.7). Since the HPLC method is able to separate AMP and IMP, reconstitution experiments were again undertaken to determine whether the HPLC could follow this reaction as well. A reaction mixture was prepared, AMP formed, and an AMP deaminase was added to the reaction mixture. Samples were again removed and, as shown in Figure 10.10, the addition of the AMP deaminase resulted in the conversion of AMP to IMP. Thus, this reaction sequence also can be followed. 

Role of purine nucleotides in muscle energy metabolism. The conversion of AMP to IMP prevents loss of adenosine from the cell. 

The purine nucleotide cycle of muscle consists of the conversion of AMP —> IMP AMP and requires AMP deaminase, adenylosuccinate synthetase, and adenylosuccinate lyase (Figure 27-24). Flux through this cycle increases during exercise. Several mechanisms have been proposed to explain how the increase in flux is responsible for the maintenance of appropriate energy levels during exercise (Chapter 21). 

Synthesis of AMP and GMP. This figure shows the pathways of IMP conversion to AMP or GMP. The number of each reaction corresponds to enzymes listed in the figure. 

Howard and Miles (1965) have described the enzymic synthesis of inosine-6- 0, inosine 5 -phosphate-6- 0, and guanosine-6- 0. In the course of this work they used infrared spectroscopy of Dj 0 and Dj 0 solutions to evaluate the conversion of 5 -AMP to 5 -IMP and the conversion of 2,6-diamino-9-/S-D-ribofuranosylpurine to guanosine. Kinetic plots of the data for these reactions were made in the same way as already indicated in Fig. 15.5 of Howard and Miles (1964). Figure 15.6 shows spectra for the enzymic conversion of 2,6-diamino-9-) -D-ribofuranosylpurine to guanosine, and Fig. 15.7 shows a plot of the absorbances of certain specific absorption bands during the course of the reaction. 

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