Adenylosuccinate Synthetase Activity Research Articles

Vitamin B6 inhibits activity of Helicobacter pylori adenylosuccinate synthetase and growth of reference and clinical, antibiotic-resistant H. pylori strains

The current therapies against gastric pathogen Helicobacter pylori are ineffective in over 20% of patients. Enzymes belonging to the purine salvage pathway are considered as novel drug targets in this pathogen. Therefore, the main aim of the current study was to determine the antibacterial activity of pyridoxal 5’-phosphate (PLP), an active form of vitamin B6, against reference and clinical strains of H. pylori. Using a broad set of microbiological, physicochemical (UV absorption, LC-MS, X-ray analysis) and in silico experiments, we were able to prove that PLP inhibits adenylosuccinate synthetase (AdSS) from H. pylori by the competition with GTP (IC50 eq ∼30 nM). This behaviour was attributed to formation of a Schiff base with a lysine residue (a covalent bond with Lys322 in the GTP binding site of AdSS) and was potentiated by the presence of vitamin C. This antibacterial activity of PLP gives hope for its future use against H. pylori.

Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering

Adenylosuccinate synthetase (EC. 6.3.4.4) encoded by purA in Bacillus subtilis, catalyzing the first step of the conversion of IMP to AMP, plays an important role in flux distribution in the purine biosynthetic pathway. In this study, we described the use of site saturation mutagenesis to obtain a desired enzyme activity of adenylosuccinate synthetase and its application in flux regulation. Based on sequence alignment and structural modeling, a library of enzyme variants was created by a semi-rational evolution strategy in position Thr238 and Pro242. Other than purA deletion, the leaky mutation purAP242N partially reduced the flux towards AMP derived from IMP and increased the riboflavin synthesis precursor GTP, while also kept the requirement of ATP synthesis for cell growth. PurAP242N was introduced into an inosine-producing strain and resulted in an approximately 4.66-fold increase in inosine production, from 0.088±0.009g/L to 0.41±0.051g/L, in minimal medium without hypoxanthine accumulation. These results underline that the directed evolution of adenylosuccinate synthetase could tailor its activities and adjust metabolic flux. This mutation may provide a promising application in purine-based product accumulation, like inosine, guanosine and folate which are directly stemming from purine pathway in B. subtilis.

Effects of 3-Hydrogenkwadaphnin on intracellular purine nucleotide contents and their link to K562 cell death

Our previous studies have classified 3-Hydrogenkwadaphnin (3-HK), isolated from Dendrostellera lessertii (Thymelaeaceae), as a strong anti-proliferative agent that induces apoptosis among the treated leukemia cells mainly through IMP dehydrogenase inhibition. To disclose this point, we measured the intracellular ATP and GTP levels among K562 cells exposed to a single dose of 3-HK (7, 12 and 25nM). The HPLC analyses of the 3-HK-treated cell lysates indicated a dose-dependent reduction in both GTP and ATP levels. The reduction of purine nucleotide levels was also associated with decreased activity of adenylosuccinate synthetase (AdSS) and enhanced level of cell death through apoptosis. However, the inhibitory effect of 3-HK was reversed by exogenous addition of guanosine with respect to GTP and partially with respect to ATP level. These data suggest that 3-HK targets a delicate regulatory system which governs the life/death fate of the cells partially through modulation of the GTP/ATP pool sizes.

Nucleotide mutations in purA gene and pur operon promoter discovered in guanosine- and inosine-producing Bacillus subtilis strains

The promoter region of the pur operon, which contains 12 genes for inosine monophosphate biosynthesis from phosphoribosylpyrophosphate, and the purA gene, encoding the adenylosuccinate synthetase, were compared among wild-type and three purine-producing Bacillus subtilis strains. A single nucleotide deletion at position 55 (relative to translation start site) in purA gene was found in a high inosine-producing strain and in a high guanosine-producing strain, which correlates with the absence of adenylosuccinate synthetase activity in these strains. Within the pur operon promoter of high guanosine-producing strain, in addition to a single nucleotide deletion in PurBox1 and a single nucleotide substitution in PurBox2, there were 4 substitutions in the flanking region of the PurBoxes and 32 nucleotide mutations in the 5' untranslated region. These mutations may explain the purine accumulation in purine-producing strains and be helpful to the rational design of high-yield recombinant strains.

Purine nucleotide cycle in rat renal cortex and medulla under conditions that mimic normal and low oxygen supply

The distinctive feature of the renal function and metabolism implicate a possibility of excessive ATP degradation during insufficient oxygen supply. Protection of the purine ring against degradation is one among other functions of the purine nucleotide cycle (PNC). The purpose of this study was to estimate the activity of PNC in cytosol of rat renal cortex and medulla under conditions that mimic normal and low oxygen supply in vivo. In normoxic-like condition the rate of AMP deamination was 1.7 and 2.0 nmol/mg protein/min in the cytosol of cortex and medulla, respectively. Under this condition, the rate of IMP reamination was similar to that of AMP deamination. In a hypoxia-like condition the rate of AMP deamination increased by 41% in cytosol from both parts of the kidney, while the rate of IMP reamination remained unchanged in the cytosol of medulla and decreased by 46% in the cortex cytosol. Distribution of the other enzymes of the PNC, that is, adenylosuccinate synthetase and adenylosuccinate lyase, in the cytosol of cortex and medulla correlated with that observed for AMP deamination and IMP reamination potentials. At 150 microM IMP, the activity of adenylosuccinate synthetase in the cortex and medulla was 0.34 and 1.24 nmol/mg protein/min, respectively. Activity of the adenylosuccinate lyase was severalfold greater than the respective activity of the adenylosuccinate synthetase. These results show that the efficiency of PNC is about twice as high in the medulla cytosol as in the cortex cytosol, and that the activity of PNC in kidney is mainly limited by the activity of adenylosuccinate synthetase and supply of AMP.

Occurrence of the purine nucleotide cycle in rat pancreatic islets.

The presence of the purine nucleotide cycle is investigated in rat pancreatic islets. Adenylosuccinase, adenylate deaminase, and adenylosuccinate synthetase activities are characterized in islet homogenates. In the assay of the latter enzyme, evidence is obtained for operation of the full cycle in islet extracts. The activities of the three enzymes are not vastly different in islet and brain. These findings are discussed in the light of the role currently ascribed to the purine nucleotide cycle in producing ammonia from amino acids, in adjusting the concentration of Krebs cycle intermediates, in regulating the relative concentrations of ATP, ADP, and AMP, and in controlling the activity of phosphofructokinase.

Imbalance of purine nucleotides in alanosine-resistant baby hamster kidney cells

Human DNA was used to transform adenosine kinase (AK)-deficient BHK cells followed by selection of AK+ cells in medium containing alanosine, adenosine, and uridine (AAU medium). Twenty AAUr isolates were analyzed, and none of them contained AK activity. Several purine salvage enzymes were, however, found to be affected in these cells. The levels of hypoxanthine-guanine phosphoribosyltransferase and adenylosuccinate synthetase activities were elevated, while the adenylosuccinase activity was reduced. AAU-resistance may be explained by elevated activity of adenylosuccinate synthetase to overcome the alanosine block; thus AAUr cells were able to convert exogenous adenosine----inosine----hypoxanthine----IMP----AMPS----AMP. Moreover, these AAUr cells required exogenous purines for growth. HPLC analyses of endogenous nucleotide pools of AAUr cells showed that the levels of adenine nucleotides have diminished to less than 10% of the parental levels. These results suggest that the AAU-resistant mutation, which elicits pleiotropic phenotypes in BHK cells, affects an important component in the regulation of adenine nucleotide synthesis. By including erthyro-9-(2-hydroxy-3-nonyl)adenine in the AAU medium (renamed as AAUE medium) to block deamination of adenosine, AK+ BHK cells were isolated.

Mechanism of adenosine production by xanthine-requiring mutants derived from a Bacillus strain

The xanthine-requiring mutants defective in adenine deaminase (adenase) derived from a Bacillus strain accumulate much adenosine. The mechanism of adenosine production was investigated. Limitation of the guanine-related substances in the fermentation medium facilitated the adenosine accumulation, but the excess of those suppressed it. Metabolic regulation of the purine nucleotide biosynthesis was supposed to be released from both feedback inhibition and repression by limiting the concentration of guanine-related substances in the cells caused by xanthine-requirement. Deficiency in the deaminase activities of adenine, adenosine and AMP and the weak adenosine phosphorylase activity contributed to adenosine accumulation. No apparent changes were observed in the adenylosuccinate synthetase activity and the dephosphorylation activity of AMP compared with the wild strain.

STUDIES OF PURINE BIOSYNTHESIS IN CHINESEHAMSTER CELL MDTANTS DEFICIENT IN ADENYLOSUCCINASE: 108

Purine biosynthesis and growth rate studies were carried out on a mutant of Chinese Hamster Ovary Cells (CHO-Ade I) deficient in the adenylate biosynthetic enzyme Adenylosuccinase (ASase). The ratio of the rate of adenylate to guanylate synthesis are altered in CHO-Ade I (3:88) compared to the nearly equal distribution of these nucleotides (49:51) in the wild type parent line CHO-K1. The decrease in the ratio of adenylates to guanylates results from an apparent stimulation in the rate of guanylate synthesis in addition to the expected reduced rate of adenylate synthesis. In comparison to the wild type CHO cells the ASase deficient cells show an increased accumulation of hypoxanthine-containing compounds in both the cell and media (excreted) fractions. CHO-Ade I cells display a marked elevation in the activity of the branch point enzyme adenylosuccinate synthetase suggesting a direct regulation of this enzyme by either adenylosuccinate or the ultimate product adenylates. Similar to patients with a deficiency of this enzyme SAICA riboside and succinyladenosine are excreted into the medium by CHO-Ade I cells.

Alterations of inosinate branchpoint enzymes in cultured human lymphoblasts

The specific activities of the three enzymes of the inosinate branchpoint are independently regulated when lymphoblasts are grown under various tissue culture conditions. In comparison to rapidly dividing cells, lymphoblasts at high cell density with no cellular division have decreased activity of the enzymes which commit inosinate to adenylate or guanylate, while cytoplasmic 5′-nucleotidase is relatively preserved. A linear relationship between inosinate dehydrogenase activity and growth rate ( r = 0.92) exists in lymphoblasts with slowed growth rates. In contrast, in dividing cells adenylosuccinate synthetase and 5′-nucleotidase do not vary with growth rate. Adenylosuccinate synthetase and inosinate dehydrogenase activities appear to be related to the presence or rate of cellular division, as opposed to the presence or degree of neoplastic transformation. Lymphoblast lines with alterations of specific purine metabolic enzymes have characteristic alteration of the inosinate utilizing enzymes. Deficiencies of purine nucleoside phosphorylase or hypoxanthine phosphoribisyltransferase, abnormalities which render the cell unable to salvage purine effectively, are associated with depressed inosinate dehydrogenase activity. Insertion of the hypoxanthine phosphoribosyltransferase gene into hypoxanthine phosphoribosyltransferase-deficient cells normalizes inosinate dehydrogenase activity, while a hypoxanthine phosphoribosyltransferase-deficient mutant selected from a hypoxanthine phosphoribosyltransferase-containing line has depressed inosinate dehydrogenase activity. In contrast, overactivity of phosphoribosylpyrophosphate synthetase, with enhanced excretion of purines due to excessive production, is associated with elevated inosinate dehydrogenase activity. Inosinate dehydrogenase appears to be regulated according to the availability of purine nucleotides. Patients who overproduce uric acid and potentially have undescribed purine metabolic defects are now being screened for abnormalities in the inosinate branchpoint enzymes.

Metabolism and salvage of adenine and hypoxanthine by myocytes isolated from mature rat heart

Adenine and hypoxanthine can be utilised by cardiac muscle cells as substrates for the synthesis of ATP. A possible therapeutic advantage of these compounds as high-energy precursors is their lack of vasoactive properties. Myocytes isolated from mature rat heart have been used to establish in kinetic detail the capacity of the heart to incorporate adenine, hypoxanthine and ribose into cellular nucleotides. Maximum rates of catalysis by enzymes on the salvage pathways have been established. Whilst the rate of incorporation of adenine into the ATP pool appears to depend upon intracellular concentrations of adenine and phosphoribosylpyrophosphate, for hypoxanthine the pattern is more complex. Hypoxanthine is salvaged at a slow rate compared with adenine, and is incorporated into GTP and IMP as well as into adenine nucleotides. The rate of incorporation of hypoxanthine into both IMP and ATP is accelerated in myocytes incubated with ribose. However, the rate-limiting reaction appears to be that catalysed by adenylosuccinate synthetase, for the rate of ATP synthesis is not accelerated when hypoxanthine concentration is increased from 10 to 50 μM, while the rate of IMP synthesis is more than doubled. Adenine and hypoxanthine phosphoribosyl transferases are present in equal catalytic amounts, but rat cardiac myocytes have very little adenylosuccinate synthetase activity. Exogenous ribose is incorporated into adenine nucleotides in amounts equimolar with adenine or hypoxanthine.

Intermediary purine-metabolizing enzymes from the cytosol of Dictyostelium discoideum monitored by high-performance liquid chromatography

The use of high-performance liquid chromatography to identify and quantitate five purine-metabolizing enzymes from a partially purified subcellular fraction of the eucaroytic microorganism Dictyostelium discoideum is described. All HPLC separations were carried out in an isocratic manner using reverse-phase C 18 as the stationary phase. The mobile phase consisted of a phosphate buffer with either methanol or acetonitrile as cosolvent, and optimal separation conditions were attained by varying the organic concentration or the pH of the buffer or by employing pairedion chromatographic techniques. Substrates and products were detected at either 254 nm for the purines or 295 nm for the formycin analogs. An adenosine kinase activity was identified, and it was demonstrated that formyin A (FoA) could be substituted for adenosine as the phosphate acceptor, yielding FoAMP as the product. With FoA as the substrate an apparent K m of 18.2 μ m and an apparent V max of 32.4 mmol min −1 mg −1 were observed for the activity. A purine nucleoside phosphorylase activity was found to cleave adenosine to adenine and ribosylphosphate. FoA was not found to be a substrate for this activity due to the unusual formycin C-glycosyl bond which was not hydrolyzed by enzymes or chemically with either HCl or NaOH. An adenylate deaminase activity was found to be present in the cytosolic S-100 of cells harvested during the onset of development, and this deaminase activity was greatly stimulated by ATP. With FoAMP as the substrate, an apparent K m of 236 μ m and V max of 2.78 μmol min −1 mg −1 were observed. The deamination of FoAMP could be inhibited by the addition of the natural substrate AMP. An apparent K i value of 136 μ m was determined from initial rate data. An adenylosuccinate synthetase activity was observed to have a K m value for GTP, IMP, and aspartic acid of 23, 34, and 714 μ m, respectively. The formycin analog FoIMP was not a substrate with this activity but was a competitive inhibitor of IMP. Finally hypoxanthine-guanine phosphoribosyltransferase was found to have K m and V max values for hypoxanthine of 55.5 μ m and 34.3 nmol −1 min −1 mg 1. When guanine was used as the substrate, the rate of nucleotide formation was 50% that with hypoxanthine as the substrate. The advantages of using HPLC to examine the interconnecting activities of a multienzyme complex in subcellular fractions are discussed, including the increased sensitivity obtained by using formycin analogs in the assay procedures. The methodology is extended to interpreting a series of enzyme reactions in which the product of the first was the substrate for the second.

Adenylosuccinate synthetase from Dictyostelium discoideum: Effects of hadacidin analogs and binding of [ 14C]hadacidin

The enzyme adenylosuccinate (sAMP) synthetase has been partially purified from Dictyostelium discoideum using hadacidin-Sepharose 4B affinity chromatography, anion-exchange high-performance liquid chromatography (HPLC), and gel-filtration HPLC, resulting in a 2600-fold purification. Using a newly developed HPLC procedure to assay activity, it has been found that D. discoideum adenylosuccinate synthetase activity has apparent K m values for the substrates IMP, GTP, and aspartate of 36, 23, and 714 μ m, respectively. The analog guanosine-5′-(β,γ-imino)triphosphate was found to be an inhibitor of GTP with a K i of 15 μ m, and IMP was competitively inhibited by its analog formycin B monophosphate with a K i of 80 μ m. An analysis of these kinetic data showed a pattern consistent with a fully random terter mechanism. Hadacidin, an analog of aspartate, was an inhibitor of that substrate at 86 μ m. Other analogs of hadacidin were synthesized and examined for their effect on the sAMP synthetase activity. Compared to hadacidin, which produced 100% inhibition at 5 m m, it was observed that N-acetyl- N-hydroxyglycine, N-formylglycine, N-acetylglycine, and N-hydroxyglycine all inhibited between 50 and 75%, with N-(thiocarboxy)- l-aspartic anhydride less effective at 27%, and N-benzoylglycine at only 6%. N-Formylsarcosine, N-acetylmethionine, O-methylpyruvate oxime, and hadacidin methylester had no effect at this concentration. The adenylosuccinate synthetase activity was dependent on metal ions with maximum activity being obtained with Mg 2+. The ability of the aspartate analog hadacidin to bind to the purified adenylosuccinate synthetase was demonstrated using anion-exchange HPLC and [ formyl- 14C]hadacidin. The radioactivity coeluted with the adenylosuccinate synthetase and the bound, radiolabeled hadacidin was displaced by excess aspartate.

Genetic studies on the role of the nucleoside transport function in nucleoside efflux, the inosine cycle, and purine biosynthesis.

A mutant clone (AU-100) which is 90% deficient in adenylosuccinate synthetase activity was characterized from wild-type murine S49 T-lymphoma cells. This AU-100 cell line and its hypoxanthine-guanine phosphoribosyltransferase-deficient derivative, AUTG-50B, overproduce purines severalfold and excrete massive amounts of inosine into the culture medium (Ullman et al., Proc. Natl. Acad. Sci. U.S.A. 79:5127-5131, 1982). We introduced a mutation into both of these cell lines which make them incapable of taking up nucleosides from the culture medium. The genetic deficiency in nucleoside transport prevents the adenylosuccinate synthetase-deficient AU-100 cells from excreting inosine. Because of an extremely efficient intracellular inosine salvage system, the nucleoside transport-deficient AU-100 cells also no longer overproduce purines. AUTG-50B cells which have been made genetically deficient in nucleoside transport still overproduce purines but excrete hypoxanthine rather than inosine. These studies demonstrate genetically that nucleoside transport and nucleoside efflux share a common component and that nucleoside transport has an important regulatory function which profoundly affects the rates of purine biosynthesis and purine salvage.

Effect of acidosis and uninepherectomy on isozymes of adenylosuccinate synthetase in rat kidney

Normal rat kidney contains primarily the L isozyme of adenylosuccinate synthetase. The increase in total adenylosuccinate synthetase activity that occures in response to NH 4Cl-feeding or a low potassium diet is mainly due to increase in the L isozyme, rather than to an increase in the M isozyme. 1 day after uninephrectomy there is little change in total adenylosuccinate synthetase activity or isozyme distribution in the remaining kidney. These results do not support extension to kidney of the theory proposed for liver that the L isozyme is involved in purine biosynthesis while the M isozyme is involved in ammonia production from amino acids via the purine nucleotide cycle.

Regulation, Genetics, and Properties of Adenylosuccinate Synthetase: A Review

This chapter discusses regulation, genetics, and the properties of adenylosuccinate synthetase. Adenylosuccinate formed by adenylosuccinate synthetase is cleaved by adenylosuccinate lyase to form adenosine monophosphate (AMP). The two-step conversion of inosine 5' monophosphate (IMP) to AMP is 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. The determination of the enzymatic activity of adenylosuccinate synthetase is most easily accomplished by observing the increase in absorbance at 280 nm, accompanying the conversion of IMP to adenylosuccinate. The most versatile system for the synthetase assay utilizes endogenous or added lyase and adenylate kinase to trap the adenylosuccinate into adenosine diphosphate and adenosine monophosphate. Adenylosuccinate synthetase is subject to feedback and product inhibition by AMP, adenylosuccinate, guanosine diphosphate, and guanosine monophosphate.

Effect of unusual guanosine nucleotides on the activities of some escherichia coli cellular enzymes

Unusual guanosine nucleotides guanosine 5′-diphosphate 3′-diphosphate (ppGpp, also known as MSI) and guanosine 5′-diphosphate 3′-monophosphate (ppGp, also known as MSIII) accumulate to high concentrations in wild-type cells of Escherichia coli during amino acid starvation. We reported here that both nucleotides strongly inhibit the activity of enzymes IMP dehydrogenase and adenylosuccinate synthetase, the first enzymes of the guanylate and adenylate biosynthetic pathways. In both case, ppGp (MSIII) is a stronger inhibitor than ppGPP (MSI). On the other hand, these two nucleotides exhibited opposite effects on the activity of phospho enolpyruvate carboxylase, the enzyme that utilizes phospho enolpyruvate. At their respective physiological concentrations, the activity of phospho enolpyruvate carboxylase is activated by ppGpp and inhibited by ppGp.

The purine nucleotide cycle in the regulation of ammoniagenesis during induction and cessation of chronic acidosis in the rat kidney.

The effect of chronic acid feeding and its subsequent withdrawal was determined on the amounts of the metabolic intermediates and enzymic activities of the purine nucleotide cycle. Sprague-Dawley rats were given 1.5% (w/v) NH4Cl in their drinking water for 5 days. The renal excretion of NH3 rose 70-fold and the rats developed acidosis. The amount of renal IMP rose from a control value of 4.5 +/- 2.2 to 20.4 +/- 3.7nmol/g of kidney after 48h of acid feeding (P less than 0.001) and fell to normal within 48h of the recovery. Adenylosuccinate concentrations fell from a control value of 4.5 +/- 0.9nmol/g of kidney to 1.2 +/- 0.3nmol/g (P less than 0.005) by day 5 of acidosis and continued to fall to undetectable values by 48h after recovery. The amount of AMP remained constant through the acid-feeding and the recovery periods. The activity of adenylosuccinate synthetase, the rate-limiting enzyme of the purine nucleotide cycle, paralleled the rise and fall in NH3 excretion. The activities of phosphate-dependent glutaminase and glutamate dehydrogenase were elevated during the acid-feeding and the recovery period. Thus changes in the purine nucleotide cycle correlate with changes in NH3 excretion to a more parallel degree than does the activity of glutaminase or glutamate dehydrogenase.

A novel synergistic effect of alanosine and guanine on adenine nucleotide synthesis in mammalian cells. Alanosine as a useful probe for investigating purine nucleotide metabolism.

A novel synergistic effect of the antitumor agent alanosine (2-amino-3-(hydroxynitrosoamino) propionic acid), which specifically inhibits the enzyme adenylosuccinate synthetase (ASS) and guanine on the growth of Chinese hamster ovary (CHO) cells and human diploid fibroblasts (HDF) has been observed. In the presence of subinhibitory concentrations of alanosine, both CHO cells and the HDF show excessive sensitivity to exogenous guanine--a phenotype which closely resembles that seen with some of the mutants containing reduced enzymatic activity of ASS. The growth inhibitory effects of alanosine, or alanosine and guanine, on CHO cells are completely reverted by the addition of adenine to the culture medium, and the synergistic effect of guanine is not observed in mutants which lack the enzyme hypoxanthine-guanine phosphoribosyl transferase. These resuls suggest that guanine nucleotides exert a regulatory effect on the activity of the enzyme adenylosuccinate synthetase. The ability to confer the guanine-sensitive phenotype and its modulation by subinhibitory concentrations of alanosine in different cell types indicates that alanosine provides a useful probe for investigating the regulation of purine nucleotide metabolism in mammalian cells.

Increases in purine excretion and rate of synthesis by drugs inhibiting IMP dehydrogenase or adenylosuccinate synthetase activities.

Hershfield reported an assay system devoid of hypoxanthine for evaluating the rate of purine synthesis de novo based on rates of [14C]-formate incorporation into intracellular purine components of lymphoblasts and into purines excreted into the culture medium. Using this system normal and in vitro selected, biochemically-characterized HPRT* deficient lines were found to have similar rates of total purine synthesis; however, these cell lines differed markedly in the excretion of newly-formed purine into the medium (1,2).