Enzymes adenylosuccinate synthetase

The enzyme adenylosuccinate synthetase condenses IMP with aspartic acid to form adenylosuccinate (sAMP). GTP participates directly in the reaction process, and during the course of the reaction GDP is formed. 

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.

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... 

A three-substrate, three-product enzyme-catalyzed reaction scheme in which the three substrates (A, B, and C) and three products (P, Q, and R) can bind to and be released in any order. A number of enzymes have been reported to have this mechanism for example, adenylosuccinate synthetase , glutamate dehydrogenase, glutamine synthetase , formyltetrahydrofolate synthetase, and tubulin tyrosine ligase . See Multisubstrate Mechanisms... 

AMP is a competitive inhibitor (see "Enzymes Catalysis and Kinetics" Lecture) of Adenylosuccinate Synthetase, GMP competitively inhibits IMP Dehydrogenase. Note GTP is required for AMP syndesis and ATP is required for GMP synthesis, hence there is coordinated regulation of these nucleotides. 

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. 

AMP is also an intermediate in de novo synthesis of ATP (reaction 3 below) and salvage synthesis of ATP (reactions 4, 5, and 8 below). AMP is an allosteric activator of glycogen phosphorylase b, and phosphofructokinase, as well as an allosteric inhibitor of fructose-1,6-bisphosphatase and adenylosuccinate synthetase. AMP is also an allosteric inhibitor of glutamine synthetase, an enzyme with a central role in nitrogen metabolism in the cell. 

Leishmania adenylosuccinate synthetase has a narrow substrate specificity but accepts several IMP analogs which include allopurinol ribonucleotide (34). The GMP reductase from L. donovani is quite different from the human GMP reductase (35) and IMP analogs are more potent inhibitors for it. Other leishmanial enzymes that have been investigated include IMP dehydrogenase (36), nucleoside hydrolase and phos-phorylase activities (37,38), adenosine kinase (39), nucleotidases (40) and the adenylosuccinate lyase (34). 

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). 

The enzymes that convert IMP to XMP and adenylosuccinate are both regulated. GMP inhibits the activity of IMP dehydrogenase, and AMP inhibits adenylosuccinate synthetase. Note that the synthesis of AMP is dependent on GTP (of which GMP is a precursor), whereas the synthesis of GMP is dependent on ATP (which is made from AMP). This serves as a type of positive regulatory mechanism to balance the pools of these precursors when the levels of ATP are high, GMP will be... 

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. 

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. 

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.

A st e-specific difference in the aaivities of enzymes is another charaaeristic of some Leishmania spiecies, with promastigotes containing adenine deaminase and amastigotes containing adenosine deaminase. IMP formed in the cell can be converted to AMP by adenylosuccinate synthetase and adenylosuccinate lyase whereas XMP is converted to IMP by GMP synthetase and GMP reduaase. Moreover, IMP dehydrogenase has also the ability to convert XMP to GMP. ... 

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. 

Adenylosuccinate synthetase activity has been observed in almost all tissues examined. The only exception is the human erythrocyte (30). Davey (18) had reported that the enz3rme was absent in rabbit heart, lung, and kidney, but it was demonstrated subsequently in those tissues (31). Highest levels of enzyme activity occur in skeletal and heart muscle and the testes (24). 

It appears that adenylosuccinate synthetase is subject to feedback and product inhibition by AMP, adenylosuccinate, GDP, and GMP. The KiS reported for AMP range from 10 to 3000 fx.M for the enzyme from normal cells from various sources 34, 38, 45, 50). The for AMP for adenylosuccinate synthetase from Novikoflf ascites and Walker carcinoma 256 tumors is in the range of 150-190 fiM and are less sensitive to inhibition than the acidic isozyme from rat liver for which the/f i for AMP was 47 /xM 45). In some cases, nucleoside monophosphates such as AMP interact with both nucleotide sites, producing noncompetitive inhibition patterns 38, 45). 

Mercaptopurine ribonucleotide is an important drug that inhibits various nucleotide-metabolizing enzymes (71). With adenylosuccinate synthetase from mammalian sources, inhibition by 6-mercaptopurine ribonucleotide appears to be similar to AMP, interacting with both nucleotide substrate sites. KiS of 44-55 fiM have been reported (45). With the bacterial enzymes, somewhat lower A iS have been observed, 10-25 fj-M, and the inhibition appears to be generally competitive with respect to IMP only (9,14). 

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... 

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. 

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... 

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 substrate specificity of adenylosuccinate synthetase is strict inosi-nate cannot be replaced by hypoxanthine, inosine, or phosphoribosyl aminoimidazole carboxamide L-aspartate cannot be replaced by ammonia or other d- or L-amino acids and other nucleoside mono-, di-, and triphosphates cannot replace GTP. Divalent cations are required. Bacterial, plant, and animal enzymes have similar specificities and general properties, although Michaelis constants differ somewhat. These are approximately 10 to 10 Af for GTP and aspartate, and 3 X Iff" M for ino-sinate. 

Increased enzyme activity may be due to increased concentrations of nucleotide substrates, or by other mechanisms. For example, GTP is required as a substrate for adenylosuccinate synthetase, and ATP is required for guanylate synthetase in addition, ATP activates adenylate deaminase (33). 

---