Purines adenylosuccinate synthetase

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... 

Didanosine is a synthetic purine nucleoside analog that inhibits the activity of reverse transcriptase in HIV-1, HIV-2, other retroviruses and zidovudine-resistant strains. A nucleobase carrier helps transport it into the cell where it needs to be phosphorylated by 5 -nucleoiidase and inosine 5 -monophosphate phosphotransferase to didanosine S -monophosphate. Adenylosuccinate synthetase and adenylosuccinate lyase then convert didanosine 5 -monophosphate to dideoxyadenosine S -monophosphate, followed by its conversion to diphosphate by adenylate kinase and phosphoribosyl pyrophosphate synthetase, which is then phosphorylated by creatine kinase and phosphoribosyl pyrophosphate synthetase to dideoxyadenosine S -triphosphate, the active reverse transcriptase inhibitor. Dideoxyadenosine triphosphate inhibits the activity of HIV reverse transcriptase by competing with the natural substrate, deoxyadenosine triphosphate, and its incorporation into viral DNA causes termination of viral DNA chain elongation. It is 10-100-fold less potent than zidovudine in its antiviral activity, but is more active than zidovudine in nondividing and quiescent cells. At clinically relevant doses, it is not toxic to hematopoietic precursor cells or lymphocytes, and the resistance to the drug results from site-directed mutagenesis at codons 65 and 74 of viral reverse transcriptase. 

The enzymatic activity of amido phosphoribosyltransferase (P-Rib-PP— PR A) is low and flux through the de novo pathway in vivo is regulated by the end-products, AMP, IMP and GMP. Inhibition of reaction 1 by dihydrofolate polyglutamates would signal the unavailability of /V1()-formyl tetrahydrofolate, required as a substrate at reactions 3 and 9 of the pathway. The purine pathway is subject to further regulation at the branch point from IMP XMP is a potent inhibitor of IMP cyclohydrolase (FAICAR—> IMP), AMP inhibits adenylosuccinate synthetase (IMP—> sAMP) and GMP inhibits IMP dehydrogenase (IMP— XMP). 

In muscle, a unique nucleotide reutilization pathway, known as the purine nucleotide cycle, uses three enzymes myoadenylate deaminase, adenylosuccinate synthetase, and adenylosuccinate lyase. In this cycle, AMP is converted to IMP with formation of NH3, and IMP is then reconverted to AMP. Myoadenylate deaminase deficiency produces a relatively benign disorder of muscle... 

Feedback regulation of the de novo pathway of purine biosynthesis. Solid lines represent metabolic pathways, and broken lines represent sites of feedback regulation. , Stimulatory effect , inhibitory effect. Regulatory enzymes A, PRPP synthetase B, amidophosphoribosyltransferase C, adenylosuccinate synthetase D, IMP dehydrogenase. 

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). 

In addition to salvaging purines, most cells interconvert adenine and guanine nucleotides. Inosine monophosphate (IMP), is the common intermediate. IMP is converted into AMP by a two-step reaction catalyzed by adenylosuccinate synthetase and adenylosuccinate lyase. Guanine nucleotides are formed in a two-step reaction in which IMP is converted into xanthine monophosphate (XMP) and then aminated to GMP. Both GMP and AMP can be reconverted into IMP. Mammalian cells can also deaminate adenosine to inosine and guanine to xanthine (Fig. 6.1). 

Individual enzymes of purine salvage are similar to those of Leishmania. PRTase activities were found for adenine, hypoxanthine, and guanine in the three forms (43). As in Leishmania, there is also a separate xanthine PRTase. Nucleoside kinase activities were found for adenosine, inosine, and guanosine (43), nucleoside hydrolase activities for inosine and guanosine and a nucleoside phosphorylase activity for adenosine. There are both nucleoside hydrolase and phosphorylase activities in epimastigotes (44,45). The adenylosuccinate synthetase and adenylosuccinate lyase are essentially identical to those found in L. donovani (46). 

FR901483 in suppressing the immune system results from an antimetabolite activity whereby adenylosuccinate synthetase and/or adenylosuccinate lyase are inhibited. These enzymes function as key catalysts in the de novo purine nucleotide biosynthetic pathway. Addition of adenosine or deoxyadenosine (but not deoxyguanosine, deoxycytidine, uridine or thymidine) results in elimination of the immunosuppressive activity of FR901483. Thus, FR901483 may inhibit one of the key steps for adenosine biosynthesis (Scheme 1). 

Fig. 41.9. The regulation of purine synthesis. PRPP synthetase has two distinct allosteric sites, one for ADP, the other for GDP. Glutamine phosphoribosyl amidotransferase contains adenine nucleotide and guanine nucleotide binding sites the monophosphates are the most important, although the di- and tri-phosphates will also bind to and inhibit the enzyme. Adenylosuccinate synthetase is inhibited by AMP IMP dehydrogenase is inhibited by GMP.

AMP synthesis (The two reactions of AMP synthesis minic steps in the purine pathway leading to IMP.) In Step 1, the 6-0 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. 

Figure 1. Purine salvage pathways of Leishmania species. Enzymes 1) phosphoribosyltransferase 2) adenine deaminase 3) guanine deaminase 4) adenosine deaminase 5) nucleoside kinase 6, nucleotidase 7) AMP deaminase 8) adenylosuccinate synthetase 9) adenylosuccinate lyase 10) AMP kinase 11) GMP kinase 12) IMP dehydrogenase 13) GMP synthetase 14) GMP reductase.

Figure 3. Compartmentalization of the purine salvage pathway of Leishmania. Abbreviations are as follows AAH, adenine aminohydrolase XPRT, xanthine phosphoribosyltransferase HGPRT, hypoxanthine-guaninephosphoribosyltransferase ADSS, adenylosuccinate synthetase ASL, adenylosuccinate lyase IMPDH, inosine monophosphate dehydrogenase GMPS, gua-nosine monophosphate synthase GDA, guanine deaminase AMPDA, adenosine monophosphate deaminase GMPR, guanosine monophosphate reductase APRT, adenine phosphoribosyltransferase AK, adenosine kinase. Enzymes that have been localized are shown in black and those that are predicted to be in the denoted locations are depicted in gray.

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. 

Reactions in which aspartate functions as a nitrogen donor have not been studied in the detail that reactions involving glutamine have. The important role of adenylosuccinate synthetase at a branch point of purine metabolism and as a component of the purine nucleotide cycle makes this enzyme a challenging subject for study. This article will deal with regulatory, kinetic, and genetic aspects of adenylosuccinate synthetase from a variety of systems. 

Since the two isozymes of adenylosuccinate synthetase differ so markedly, changes in the relative amounts of the two could drastically affect the regulation of the reaction they catalyze, and therefore the direction of purine nucleotide metabolism. Determination of this ratio could be a useful indicator of the relative importance of the biosynthetic and the cyclic aspects of the adenine nucleotide interconversion pathway in different tissues or under different metabolic conditions. 

Adenylosuccinate synthetase is subject to inhibition by the products of its reaction and by a wide variety of substrate analogs. It was shown by Wyngaarden and Greenland 34) that the enzyme from E. coli is strongly inhibited by purine nucleotides, albeit with little specificity. For example, AMP, dAMP, GMP, and dGMP all inhibited the enzyme... 

Adenylosuccinate synthetase enzymatic activity is responsive to the intracellular concentration of purine nucleotide monophosphates, but AMP-specific inhibition is probably not important. 

The regulation of mammalian adenylosuccinate synthetase is complicated. It is dependent on the isozyme content and levels in a given tissue as well as the effects of substrate and product levels. The two isozymes may have different metabolic roles either in AMP biosynthesis and interconversion, or in the functions of the purine nucleotide cycle. Most studies have considered kinetic parameters for the isolated enzyme and in only a few instances has regulation been studied in vivo. Sufficient information is available concerning the regulation of the basic isozyme in muscle to consider that enzyme in detail. Factors controlling the acidic isozyme are less clearly defined. 

Overall, from the kinetic properties of muscle adenylosuccinate synthetase reported here, it would appear that the most important regulatory factor in muscle is the availability of IMP. A positive correlation between IMP concentration and adenylosuccinate production in muscle has already been demonstrated (58). The decrease in phosphocreatine during contraction would also lead to an increase in activity. However, hydrolysis of phosphocreatine will produce a concomitant increase in the concentration of inorganic phosphate, which is as good an inhibitor of adenylosuccinate synthetase as phosphocreatine (28). The changes in GDP and fructose 1,6-bisphosphate would appear to oppose this process. Whether these two metabolites serve to dampen the oscillations of the purine nucleotide cycle in vivo is unclear. It has recently been demonstrated that muscle tissue has a significant capacity for de novo purine biosynthesis 104). This requires that the basic... 

Matsuda et al. (27) showed that the adenylosuccinate synthetase basic isozyme has a lower Km for aspartate, is more sensitive to inhibition by fructose 1,6-bisphosphate, and less sensitive to inhibition by nucleotides than the acidic isozyme. These properties could indicate that the basic isozyme is regulated coordinately with glycolysis (or gluconeogenesis) as proposed for the operation of the purine nucleotide cycle in skeletal muscle. The enzyme could also be affected by the availability of aspartate, as was found in Ehrlich ascites cells. The increase in basic isozyme activity, under conditions used in this study where the animal must rely on protein for most of its energy, is consistent with the idea that it is involved in the purine nucleotide cycle. This probably is not as an alternative to glutamate dehydrogenase in urea synthesis but is simply in amino acid catabolism. The small... 

A. The Role of S. cereWs/ee Adenylosuccinate Synthetase in the Regulation of de Novo Purine Biosynthesis... 

The evidence suggested yeast adenylosuccinate synthetase catalyzed the first committed step in AMP biosynthesis and was also involved in the repression of synthesis of the early enzymes of the pathway. The mechanism by which this repression occurs remains unknown. Woods and co-workers (115,116) have isolated several prototrophic mutants of yeast defective in control of purine synthesis that are not allelic with adel2. Genetic analysis of these mutants suggested that the regulatory function of adenylosuccinate synthetase may be affected in conjunction with as many as three other gene products (116). 

To date, no biochemical evidence exists to support this notion of a bifunctional role for adenylosuccinate synthetase. In particular, the synthesis of specific enzymes has not been correlated with the loss of regulation. Instead, the loss of regulation has been followed by measuring purine excretion or by observing adenine-insensitive accumulation and polymerization of aminoimidazole ribotide (red color formation) in cells blocked at adel or add2. In addition, adenylosuccinate synthetase from S. cerevisiae has not been purified or characterized and the... 

The dual role of adenylosuccinate synthetase in purine nucleotide interconversions and in de novo AMP biosynthesis complicates studies of its regulation. There is evidence suggesting that in wild-type cells, both the de novo and the salvage pathways play an active role in the maintenance of appropriate ATP/GTP ratios (78). 

An interesting role of adenylosuccinate synthetase as a possible regulatory protein for the control of early enzymes in the purine pathway has been revealed in studies with yeast mutants [112,112a]. The loss of the synthetase by mutation is associated with an inability of adenine to prevent the synthesis of an early intermediate. It is not yet clear whether this is due to a modification of feedback inhibition or control by repression. If the latter is the case, then at least five different enzymes would come under such a control, since the continued repression of any one would not have allowed isolation of the phenotype. 

Seegmiller who pharmacologically simulated adenylosuccinate synthetase deficiency in human cells with resulting purine overproduction and overexcretion (8). These workers indicated that increased rates of purine synthesis and excretion could also be induced pharmacologically by inhibition of IMP dehydrogenase. 

These studies with wild-type and mutant cells defective in IMP dehydrogenase and the previous data with the adenylosuccinate synthetase-deficient cell line suggest that among the clinical population with dominantly inherited hyperuricemia, patients with partial deficiencies in these enzymes exist. It is hoped that these pharmacogenetic cell culture models for overproduction hyperuricemia will lead to the initiation of a search for hyperuricemia patients with either of these deficiencies. If such patients are found it may be possible to design chemotherapeutic regimens by which effectors (inhibitors) of purine synthesis might ameliorate the overproduction of purines by the de novo pathway. 

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. 

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