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. 

The enzyme was prepared from Dictyostelium discoideum. The cells were lysed, and S-100 solutions were prepared from the lysate by differential centrifugation. Samples of this solution were used in the assay. 

The reaction mixture contained HEPES-NaOH (pH 7.5), the substrate (Ap4A), and the enzyme. After a 10-minute incubation, the reactions were 

The enzyme was purified to homogeneity from Physarum polycephalum. 

The NAD glycohydrolase (EC 3.2.2.5) in this study catalyzes the hydrolysis of NAD+ to form nicotinamide, adenosine diphosphate ribose (ADPR), and H+. The assay developed for this activity follows the disappearance of the substrate NAD+ and the production of nicotinamide. 

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It is likely that ara-HxMP similarly exerts its antiviral activity in the form of the triphosphate, ara-HxTP, since ara-HxTP inhibits HSV-1 DNA polymerase (49). Another possible explanation of the antiviral activity of ara-HxTP is that it is metaboHcaHy converted to ara-AMP. In fact, it has been shown at Wellcome Research Laboratories that ara-HxMP is a substrate for adenylosuccinate synthetase, and that the resulting arabinofuranosyladenylosuccinate is cleaved to ara-AMP by adenylosuccinate lyase (1). The selective action of ara-A against HSV appears to be a consequence of the preferential inhibition of ara-ATP against HSV-1 and HSV-2 polymerases. Ara-ATP also inhibits normal cellular DNA polymerases, which may be the reason for its cellular toxicity. Also, it has been observed that ara-A is incorporated uniformly throughout the HSV-1 genome, which may result in defective viral DNA (50). 

The conversion of inosinic acid to adenylic acid is a two-step process [65] The first step, the conversion of inosinic acid to adenylosuccinic acid, is mediated by adenylosuccinate synthetase [65], which is inhibited by 6-mercaptopurine ribonucleotide [309-311] in a non-competitive manner, although the exact nature of this inhibition is not known [67]. The second step of the sequence, the conversion of adenylosuccinic acid to adenylic acid, is also inhibited by 6-mercaptopurine ribonucleotide [66, 311], and in this case the inhibition is competitive with respect to the substrate [67]. 

Although tumour growth inhibition by 6-mercaptopurine has been attributed to the inhibition of the conversion of inosinic acid to adenylic acid [309, 310, 312], probably at the first step, this conversion by cell-free extracts from the exquisitely sensitive tumour adenocarcinoma 755 was inhibited only at high levels of 6-mercaptopurine ribonucleotide [313]. Furthermore, hadacidin (A -formylhydroxyaminoacetic acid) is an excellent inhibitor of adenylosuccinate synthetase [314, 315], and yet it has little antitumour activity and is not cytotoxic, showing that this inhibition may be relatively unimportant to cells. 

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

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... 

GDP NADH Disappearance Pyruvate Kinase and Lactate Dehydrogenase Adenylosuccinate Synthetase ... 

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 AMINOHYDROLASE ADENYLOSUCCINATE LYASE ADENYLOSUCCINATE SYNTHETASE Adenylyl cyclase,... 

ADENYLOSUCCINATE SYNTHETASE DEOXYTHYMIDINE KINASE FUCOSE-1-PHOSPHATE GUANYLYL-TRANSFERASE... 

ADENYLOSUCCINATE SYNTHETASE Phosphoryl-phosphinate adduct, d-ALANINE-d-ALANINE LIGASE PHOSPHOSERINE AMINOTRANSEERASE... 

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. 

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). 

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 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. 

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). 

Up until 2003, evidence for the presence of adenylosuccinate synthetase, which catalyses the condensation of IMP with aspartate to form adenylosuccinate and is the first committed step in the synthesis of AMP or GMP from IMP, has relied on the inhibitory action of hadacidin, an aspartate analogue (Webster et al., 1984a). But, in 2002, the dimeric... 

Eaazhisai, K., Jayalakshmi, R., Gayathri, P., Anand, R. P., Sumathy, K., Balaram, H., and Murthy, M. R. (2004). Crystal structure of fully ligated adenylosuccinate synthetase from Plasmodium falciparum. ]. Mol. Biol. 335,1251-1264. 

Jayalakshmi, R., Sumathy, K., and Balaram, H. (2002). Purification and characterization of recombinant Plasmodium falciparum adenylosuccinate synthetase expressed in Escherichia coli. Protein Expr. Purif. 25, 65-72. 

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. 

IMP is a branch point between synthesis of GMP and AMP (Figure 22.6). IMP is acted upon by adenylosuccinate synthetase in AMP biosynthesis and by IMP dehydrogenase in GMP biosynthesis. 

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). 

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