Adenylosuccinate synthetase reaction

The adenylosuccinate synthetase reaction poses an interesting and general problem regarding the synthesis of carbon-nitrogen bonds coupled to nucleoside triphosphate hydrolysis. The three alternative mechanisms shown in Fig. 2 have been proposed for adenylosuccinate... 

Adenylosuccinate synthetase (AMPSase EC 6.3.4.4) catalyzes the following reversible reaction ... 

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... 

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). 

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. 

Figure 9.112 Separation of substrates and products of reaction catalyzed by adenylosuccinate synthetase. Column Prepacked C18 /xBondapak, 10 /im particle size. Mobile phase 65 mM potassium phosphate, 1 mM tetrabutylammonium phosphate, 10% methanol at pH 4.4. Absorbance was measured at 254 nm. (From Rossomando, 1987.)...

Figure 9.113 HPLC elution profiles of adenylosuccinate synthetase incubation mixtures. This reaction was initiated by the addition of 1.25 /xmol of aspartate (pH 7.4). At 5-minute intervals, 20 /xL samples were injected onto the HPLC reversed-phase column and eluted. Inset Time-dependent utilization of IMP and the formation of sAMP, as determined by integration of the respective peaks from the HPLC chromatograms. (From Jahngen and Rossomando, 1984.)...

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. 

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). 

IMP serves as the branchpoint from which both adenine and guanine nucleotides can be produced (see Fig. 41.2). Adenosine monophosphate (AMP) is derived from IMP in two steps (Fig. 41.7). In the first step, aspartate is added to IMP to form adenylosuccinate, a reaction similar to the one catalyzed by argininosuccinate synthetase in the urea cycle. Note how this reaction requires a high-energy bond, donated by GTP Fumarate is then released from the adenylosuccinate by the enzyme adenylosuccinase to form AMP. 

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. 

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... 

In bone marrow extracts, IMP served as a precursor of both AMP and GMP (148-150). The transformation of IMP to AMP by the marrow enzyme system was dependent upon the addition of L-aspartic acid and a high energy phosphate source. Adenylosuccinic acid (Fig. 12), which was isolated and identified at the same time, was established as the intermediate in the formation of AMP from IMP. (13S, 136,151,158) The formation of adenylosuccinic acid [Eq. (15)] required GTP specifically and was studied extensively in E. coli (133) the enzyme catalyzing this reaction was named adenylosuccinate synthetase. 

Figure 10.7 Schematic representation of the formation and fate of IMP. Formation of IMP is catalyzed by the enzyme hypoxanthine/guanine phosphoribosyl tranfer-ase (1) from the substrate hypoxanthine (Hypo) and phosphoribosyl pyrophosphate (PRibPP). IMP is shown undergoing several reactions the first (2) is catalyzed by 5 -nucleotidase to form inosine (INO) and orthophosphate (Pj) the other (3) is a two-step reaction catalyzed by sAMP synthetase to form adenylosuccinate (sAMP) and (4) by the enzyme sAMP lyase to convert sAMP to AMP and fumarate. Finally, (3) the deamination of AMP to IMP and NHa is catalyzed by AMP deaminase.

Adenelosuccinate synthetase catalyzes the following reaction in the de novo biosynthesis of AMP IMP + Aspartic Acid + GTP <=> Adenylosuccinate + GDP + Pi... 

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