A comprehensive method for determining cellular uptake of purine nucleoside phosphorylase and adenylosuccinate synthetase inhibitors by H. pylori.

Due to the growing number of Helicobacter pylori strains resistant to currently available antibiotics, there is an urgent need to design new drugs utilizing different molecular mechanisms than those that have been used up to now. Enzymes of the purine salvage pathway are possible targets of such new antibiotics because H. pylori is not able to synthetize purine nucleotides de novo. The bacterium's recovery of purines and purine nucleotides from the environment is the only source of these essential DNA and RNA building blocks. We have identified formycins and hadacidin as potent inhibitors of purine nucleoside phosphorylase (PNP) and adenylosuccinate synthetase (AdSS) from H. pylori - two key enzymes of the purine salvage pathway. However, we have found that these compounds are not effective in H. pylori cell cultures. To address this issue, we have developed a universal comprehensive method for assessing H. pylori cell penetration by drug candidates, with three alternative detection assays. These include liquid chromatography tandem mass spectrometry, UV absorption, and inhibition of the target enzyme by the tested compound. Using this approach, we have shown that cellular uptake by H. pylori of formycins and hadacidin is very poor, which reveals why their in vitro inhibition of PNP and AdSS and their effect on H. pylori cell cultures are so different. The cell penetration assessment method developed here will be extremely useful for validating the cellular uptake of other drug candidates, facilitating the design of new potent therapeutic agents against H. pylori. KEY POINTS: • A method for assessing H. pylori cells penetration by drug candidates is described. • Three alternative detection assays that complement each other can be used. • The method may be adapted for other bacteria as well.

In the quest for new targets for pathogen eradication: the adenylosuccinate synthetase from the bacterium Helicobacter pylori.

Adenylosuccinate synthetase (AdSS) is an enzyme at regulatory point of purine metabolism. In pathogenic organisms which utilise only the purine salvage pathway, AdSS asserts itself as a promising drug target. One of these organisms is Helicobacter pylori, a wide-spread human pathogen involved in the development of many diseases. The rate of H. pylori antibiotic resistance is on the increase, making the quest for new drugs against this pathogen more important than ever. In this context, we describe here the properties of H. pylori AdSS. This enzyme exists in a dimeric active form independently of the presence of its ligands. Its narrow stability range and pH-neutral optimal working conditions reflect the bacterium's high level of adaptation to its living environment. Efficient inhibition of H. pylori AdSS with hadacidin and adenylosuccinate gives hope of finding novel drugs that aim at eradicating this dangerous pathogen.

Adenylosuccinate Is an Insulin Secretagogue Derived from Glucose-Induced Purine Metabolism.

Pancreatic islet failure, involving loss of glucose-stimulated insulin secretion (GSIS) from islet ß cells, heralds the onset of type 2 diabetes (T2D). To search for mediators of GSIS, we performed metabolomics profiling of the insulinoma cell line 832/13 and uncovered significant glucose-induced changes in purine pathway intermediates, including a decrease in inosine monophosphate (IMP) and an increase in adenylosuccinate (S-AMP), suggesting a regulatory role for the enzyme that links the two metabolites, adenylosuccinate synthase (ADSS). Inhibition of ADSS or a more proximal enzyme in the S-AMP biosynthesis pathway, adenylosuccinate lyase, lowers S-AMP levels and impairs GSIS. Addition of S-AMP to the interior of patch-clamped human ß cells amplifies exocytosis, an effect dependent upon expression of sentrin/SUMO-specific protease 1 (SENP1). S-AMP also overcomes the defect in glucose-induced exocytosis in ß cells from a human donor with T2D. S-AMP is, thus, an insulin secretagogue capable of reversing ß cell dysfunction in T2D.

Evaluation of hadacidin analogues.

Several derivatives of hadacidin have been developed and evaluated for activity against adenylosuccinate synthetase.

Adenine metabolism in Plasmodium falciparum.

Plasmodium falciparum lacks the de novo purine biosynthesis pathway and relies entirely on the salvage pathway to meet its purine nucleotide requirements. The entire flux for purine nucleotide biosynthesis in the parasite is believed to be through hypoxanthine guanine phosphoribosyltransferase (HGPRT), with the enzymes, adenosine kinase and adenine phosphoribosyltransferase (APRT) being unannotated in the Plasmodium genome database. This manuscript reports on the studies carried out to explore bypass mechanisms, if any, for AMP synthesis in the intraerythrocyitc stages of the parasite life cycle. Uptake and subsequent incorporation of radiolabel adenine in the nucleotide pool of saponin released erythrocyte free parasites implicated the role of parasite encoded enzymes in adenine metabolism. To explore the route for AMP synthesis in the parasite, we have monitored adenine mediated supplementation of metabolic viability in saponin released hadacidin (N-formyl-N-hydroxyglycine) treated parasites. Our results implicate the role of an APRT like activity that enables parasite survival when the flux through the HGPRT pathway is blocked.

A mathematical model for the adenylosuccinate synthetase reaction involved in purine biosynthesis.

BACKGROUND: Development of the mathematical models that adequately describe biochemical reactions and molecular-genetic mechanisms is one of the most important tasks in modern bioinformatics. Because the enzyme adenylosuccinate synthetase (AdSS) has long been extensively studied, a wealth of kinetic data has been accumulated. RESULTS: We describe a mathematical model for the reaction catalyzed by AdSS. The model's parameters were fitted to experimental data obtained from published literature. The advantage of our model is that it includes relationships between the reaction rate, the concentrations of three substrates (GTP, IMP and ASP), the effects of five inhibitors (GMP, GDP, AMP, ASUC and SUCC), and the influence of Mg2+ ions. CONCLUSION: Our model describes the reaction catalyzed by AdSS as a fully random process. The model structure implies that each of the inhibitors included in it is only competitive to one of the substrates. The model was tested for adequacy using experimental data published elsewhere. The values obtained for the parameters are as follows: Vmax = 1.35.10-3 mM/min, KmGTP = 0.023 mM, KmIMP = 0.02 mM, KmASP = 0.3 mM, KiGMP = 0.024 mM, KiGDP = 8.10-3 mM, KiAMP = 0.01 mM, KiASUC = 7.5.10-3 mM, KiSUCC = 8 mM, KmMg = 0.08 mM.

Unique kinetic mechanism of Plasmodium falciparum adenylosuccinate synthetase.

Adenylosuccinate synthetase (AdSS) catalyses the Mg(2+) dependent formation of adenylosuccinate from IMP and aspartate, the reaction being driven by the hydrolysis of GTP to GDP. All characterized AdSS thus far exhibit a random kinetic mechanism. We present here kinetic evidence that unlike all other AdSS, Plasmodium falciparum AdSS (PfAdSS) has ordered substrate binding. Inhibition studies show that binding of GTP requires IMP binding while aspartate binds to the enzyme-IMP-GTP complex. A structural basis for this difference in mechanism is presented. Kinetically, PfAdSS is closer to the mouse acidic isozyme rather than to the mouse basic isozyme. The mouse acidic isozyme is thought to play a role in the purine nucleotide biosynthetic pathway. Regulation of PfAdSS in vivo can therefore, be expected to be similar to the mouse acidic isozyme, in agreement with the role of PfAdSS as the only pathway for the synthesis of adenine nucleotides in the parasite. However, PfAdSS differs from both the mammalian homologs in that fructose-1,6-bisphosphate, a potent inhibitor of the mammalian enzyme, is an activator of PfAdSS. The differences highlighted here are promising in terms of species-specific drug design, targeting this essential enzyme in the parasite.

Feedback inhibition and product complexes of recombinant mouse muscle adenylosuccinate synthetase.

Adenylosuccinate synthetase governs the committed step of AMP biosynthesis, the generation of 6-phosphoryl-IMP from GTP and IMP followed by the formation of adenylosuccinate from 6-phosphoryl-IMP and l-aspartate. The enzyme is subject to feedback inhibition by AMP and adenylosuccinate, but crystallographic complexes of the mouse muscle synthetase presented here infer mechanisms of inhibition that involve potentially synergistic ligand combinations. AMP alone adopts the productive binding mode of IMP and yet stabilizes the active site in a conformation that favors the binding of Mg(2+)-IMP to the GTP pocket. On the other hand, AMP, in the presence of GDP, orthophosphate, and Mg(2+), adopts the binding mode of adenylosuccinate. Depending on circumstances then, AMP behaves as an analogue of IMP or as an analogue of adenylosuccinate. The complex of adenylosuccinate.GDP.Mg(2+).sulfate, the first structure of an adenylosuccinate-bound synthetase, reveals significant geometric distortions and tight nonbonded contacts relevant to the proposed catalytic mechanism. Adenylosuccinate forms from 6-phosphoryl-IMP and l-aspartate by the movement of the purine ring into the alpha-amino group of l-aspartate.

Ribofuranosyl triazolone: a natural product herbicide with activity on adenylosuccinate synthetase following phosphorylation.

2,4-Dihydro-4-(beta-D-ribofuranosyl)-1,2,4(3H)-triazol-3-one (2) was identified as the principal phytotoxic component of a fermentation broth derived from an Actinomadura. The compound is a new natural product, but known by synthesis. Broad-spectrum herbicidal activity was demonstrated in greenhouse tests. Metabolite reversal studies suggested the target site was adenylosuccinate synthetase, which was confirmed by direct measurement of the activity of the 5'-phosphorylated derivative on the isolated enzyme.

8-(4-Bromo-2,3-dioxobutylthio)guanosine 5'-triphosphate: a new affinity label for purine nucleotide sites in proteins.

A new affinity label, 8-(4-bromo-2,3-dioxobutylthio)guanosine 5'-triphosphate (8-BDB-TGTP), has been synthesized by initial reaction of GTP to form 8-Br-GTP, followed by its conversion to 8-thio-GTP, and finally coupling with 1,4-dibromobutanedione to produce 8-BDB-TGTP. 8-BDB-TGTP and its synthetic intermediates were characterized by thin-layer chromatography, UV, (31)P NMR spectroscopy, as well as by bromide and phosphorus analysis. Escherichia coli adenylosuccinate synthetase is inactivated by 8-BDB-TGTP at pH 7.0 at 25 degrees C. Pretreatment of the enzyme with N-ethylmaleimide (NEM) blocks the exposed Cys(291) and leads to simple pseudo-first-order kinetics of inactivation. The inactivation exhibits a nonlinear relationship of initial inactivation rate versus 8-BDB-TGTP concentration, indicating the reversible association of 8-BDB-TGTP with the enzyme prior to the formation of a covalent bond. The inactivation kinetics exhibit an apparent K(I) of 115 microM and a k(max) of 0.0262 min(-1). Reaction of the NEM-treated adenylosuccinate synthetase with 8-BDB-[(3)H]TGTP results in 1 mol of reagent incorporated/mol of enzyme subunit. Adenylosuccinate or IMP plus GTP completely protects the enzyme against 8-BDB-TGTP inactivation, whereas IMP or GTP alone provide partial protection against inactivation. AMP is much less effective in protection. The results of ligand protection studies suggest that E. coli adenylosuccinate synthetase may accommodate 8-BDB-TGTP as a GTP analog. The new affinity label may be useful for identifying catalytic amino acid residues of protein proximal to the guanosine ring.

Adenylosuccinate synthase from Saccharomyces cerevisiae: homologous overexpression, purification and characterization of the recombinant protein.

Adenylosuccinate synthase (EC 6.3.4.4) catalyses the first committed step in the synthesis of adenosine. We have overexpressed the cloned gene of Saccharomyces cerevisiae (ADE12) in S. cerevisiae. The recombinant enzyme exhibits similar kinetic behaviour to that of the native enzyme purified from S. cerevisiae. This ter-reactant dimeric enzyme shows Michaelis-Menten kinetics only with IMP. l-Aspartate and GTP display a weak negative co-operativity (Hill coefficient 0. 8-0.9). This negative co-operativity has not yet been reported for adenylosuccinate synthases from other organisms. Another unusual feature of the enzyme from S. cerevisiae is its negligible inhibition by adenine nucleotides and its pronounced inhibition by Cl(-) ions.

Implication of arginine-131 and arginine-303 in the substrate site of adenylosuccinate synthetase of Escherichia coli by affinity labeling with 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate.

Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate (6-BDB-TAMP) at pH 7.0 and 25 degrees C. The initial fast-phase inactivation is not affected by the presence of active-site ligands and can be completely eliminated by blocking Cys291 of the enzyme with N-ethylmaleimide (NEM). Reaction of the NEM-treated enzyme with 6-BDB-[32P]TAMP results in 2 mol of reagent incorporated/mol of enzyme subunit. The inactivation kinetics of the slow-phase exhibit an apparent KI of 40.6 microM and kmax of 0.0228 min-1. Active-site ligands, either adenylosuccinate or IMP and GTP, completely prevent inactivation of the enzyme by 6-BDB-TAMP, whereas IMP or IMP and aspartate is much less effective in protection. 6-BDB-TAMP-inactivated enzyme has a 3-fold increase in Km for aspartate with no change in Km for IMP or GTP. Protease digestion of 6-BDB-[32P]TAMP inactivated enzyme reveals that both Arg131 and Arg303 are modified by the affinity-labeling reagent. The crystal structure [Poland, B. W., Fromm, H. J., and Honzatko, R. B. (1996) J. Mol. Biol. 264, 1013-1027] and site-directed mutagenesis [Kang, C., Sun, N., Poland, B. W., Gorrell, A., and Fromm, H. J. (1997) J. Biol. Chem. 272, 11881-11885] of E. coli adenylosuccinate synthetase show that Arg303 interacts with the carboxyl group of aspartate and the 2'-OH of the ribose of IMP and Arg131 is involved in stabilizing aspartate in the active site of the enzyme. We conclude that 6-BDB-TAMP functions as a reactive adenylosuccinate analogue in modifying both Arg131 and Arg303 in the active site of adenylosuccinate synthetase.

Adenylosuccinate synthetase from maize. Purification, properties, and mechanism of inhibition by 5'-phosphohydantocidin.

Adenylosuccinate synthetase (AdSS) is the site of action hydantocidin, a potent microbial phytotoxin. A kinetic analysis of the mode of inhibition of a plant adenylosuccinate synthetase by the active metabolite 5'-phosphohydantocidin (5'-PH) was the objective of the present study. AdSS was purified 5800-fold from maize (Zea mays), to our knowledge the first purification of the enzyme from a plant source. N-terminal sequencing established the cleavage site of the previously published deduced sequence of the initial transcript. The subunit molecular mass was determined to be 48 kD and the isoelectric point was at pH 6.1. Values of the Michaelis constant for the three substrates IMP, GTP, and aspartate were 21, 16, and 335 microM, respectively. Inhibition of AdSS by 5'-PH was measurably time-dependent. The trace of the inactivation curve could not be altered by preincubating the enzyme and inhibitor in the absence of substrates but could be linearized by preincubating the enzyme with inhibitor, aspartate, GTP (or GDP), and inorganic phosphate. Inhibition of AdSS by 5'-PH was competitive with IMP, with an apparent Ki of 22 nM. Apparently, 5'-PH inhibits the enzyme by binding to the IMP site and forming a tight, dead-end complex.

Involvement of arginine 143 in nucleotide substrate binding at the active site of adenylosuccinate synthetase from Escherichia coli.

Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thio]phosphate (GMPSBDB) at pH 7.1 and 25 degrees C. Reaction of the enzyme with [8-3H]GMPSBDB results in the incorporation of 2 mol of the reagent/mol of subunit; in the presence of active site ligands the incorporation is reduced to 1 mol of reagent/mol of subunit. GMPSBDB reacts with Cys-291 in the initial rapid reaction which is accompanied by loss of 50% of the enzymatic activity; this reaction is not affected by the presence of active site ligands. In the slower reaction, GMPSBDB inactivates the enzyme by reacting with Arg-143. The inactivation kinetics of the slower phase are consistent with the formation of an enzyme--GMPSBDB complex having a Kd of 42 microM. Active site nucleotides, either adenylosuccinate or IMP + GTP, prevent both slower phase inactivation and labeling of Arg-143. Replacement of Arg-143 with a Leu by site-directed mutagenesis does not change the catalytic constant or the K(m) for aspartate but does significantly impair nucleotide binding: the Michaelis constants for IMP and GTP increase by 60-fold and 10-fold, respectively, in the R143L mutant. The crystal structure of the E. coli enzyme [Poland, B.W., Silva, M.M., Serra, M.A., Cho, Y., Kim, K. H., Harris, E.M.S., & Honzatko, R.B. (1993) J. Biol. Chem. 268, 25334--25342] shows that Arg-143 from one subunit projects into the putative active site of the other subunit. These results indicate that both subunits of dimeric adenylosuccinate synthetase contribute to each active site and that Arg-143 plays an important role in nucleotide binding.

High performance liquid chromatography analysis of hypoxanthine metabolism in mouse oocyte-cumulus cell complexes: effects of purine metabolic perturbants.

This study was undertaken to examine the metabolism of hypoxanthine by mouse oocyte-cumulus cell complexes. Complexes were isolated from immature mice 48 h after priming with 5 IU eCG and culture for 3 h in medium containing 14C-hypoxanthine in the absence or presence of one of three metabolic inhibitors: alanosine, mycophenolic acid, or 6-mercaptopurine. Tissue extracts from complexes were analyzed by HPLC using either a C18 reversed-phase column (for separation of purine bases and nucleosides) or an ion exchange column (for separation of nucleotides). Most of the hypoxanthine taken up by complexes was salvaged to inosine monophosphate (IMP) and then converted to nucleotides. Metabolism favored the synthesis of adenyl nucleotides over guanyl nucleotides. No evidence of metabolism to uric acid via xanthine oxidase was encountered, and metabolism to inosine via purine nucleoside phosphorylase was negligible. A similar pattern of hypoxanthine metabolism was observed in extracts of oocytes that had been denuded after the culture period. Addition of alanosine to the culture medium significantly reduced the synthesis of adenyl nucleotides in complexes and partially shunted metabolism in the direction of guanyl nucleotides. However, neither alanosine nor another inhibitor of adenylosuccinate synthetase, hadacidin, significantly influenced the meiotic arrest maintained by hypoxanthine. Mycophenolic acid eliminated conversion of IMP to guanyl nucleotides but did not appreciably affect metabolism to other nucleotides. 6-Mercaptopurine produced an increase in the hypoxanthine-containing peaks, which was consistent with suppression of purine salvage. These results demonstrate that hypoxanthine is readily salvaged by the murine oocyte-cumulus cell complex and that the inhibitor-induced changes in metabolism are consistent with the presumed mechanism of action of each inhibitor. In addition, whereas metabolism favors conversion of IMP to adenyl nucleotides, synthesis of adenyl nucleotides by this route during the culture period is apparently not required for hypoxanthine-maintained meiotic arrest in vitro.

Evidence for an arginine residue at the substrate binding site of Escherichia coli adenylosuccinate synthetase as studied by chemical modification and site-directed mutagenesis.

Chemical modification of adenylosuccinate synthetase from Escherichia coli with phenylglyoxal resulted in an inhibition of enzyme activity with a second-order rate constant of 13.6 M-1 min-1. The substrates, GTP or IMP, partially protected the enzyme against inactivation by the chemical modification. The other substrate, aspartate, had no such effect even at a high concentration. In the presence of both IMP and GTP during the modification, nearly complete protection of the enzyme against inactivation was observed. Stoichiometry studies with [7-14C]phenylglyoxal showed that only 1 reactive arginine residue was modified by the chemical reagent and that this arginine residue could be shielded by GTP and IMP. Sequence analysis of tryptic peptides indicated that Arg147 is the site of phenylglyoxal chemical modification. This arginine has been changed to leucine by site-directed mutagenesis. The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild-type system, whereas the Km for aspartate exhibited a modest decrease as compared with the native enzyme. In addition, kcat of the R147L mutant decreased by a factor of 1.3 x 10(4). On the bases of these observations, it is suggested that Arg147 is critical for enzyme catalysis.

Chemical modification of adenylosuccinate synthetase from Escherichia coli by pyridoxal 5'-phosphate. Identification of an active site lysyl residue.

Incubation of adenylosuccinate synthetase from Escherichia coli with low concentrations of pyridoxal 5'-phosphate (PLP) resulted in a rapid loss of activity (92%), concomitant with the formation of a Schiff base. The inactivation of the enzyme by PLP is apparently first order with respect to PLP. The pseudo-first order rate constant, Kapp, showed a hyperbolic dependence on the concentration of PLP, indicating that a kinetically significant PLP.enzyme intermediate is formed during the inactivation process. Stoichiometry and peptide isolation studies showed that 2 lysine residues were modified during reaction of the enzyme with PLP. The three substrates of adenylosuccinate synthetase (GTP, IMP, and aspartate) showed different effects in their ability to protect the enzyme against PLP inactivation. Complete protection of the enzyme against inactivation can be observed only in the presence of high concentrations of GTP. One lysine residue was protected under these conditions. In contrast to GTP, addition of the other two substrates either alone or together to reaction mixtures did not render protection. Peptide mapping by digesting the enzyme with trypsin revealed that the lysine shielded by GTP is Lys140. Replacing the Lys140 with Ile140 by site-directed mutagenesis resulted in total loss of the activity. These results suggest that Lys140 may play an important role in enzymatic activity.

Inhibition of adenylosuccinate lyase by L-alanosyl-5-aminoimidazole-4-carboxylic acid ribonucleotide (alanosyl-AICOR).

L-Alanosyl-5-aminoimidazole-4-carboxylic acid ribonucleotide (alanosyl-AICOR) has been synthesized enzymatically using 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) synthetase in conjunction with 5-aminoimidazole-4-carboxylic acid ribonucleotide and L-2-amino-3-(N-hydroxy-N-nitrosoamino)propionic acid (alanosine). The product was characterized by chromatography, ultraviolet spectrum and NMR spectrum at 300 MHz. Alanosyl-AICOR was not a substrate of adenylosuccinate lyase from rat skeletal muscle, but it was an apparent competitive inhibitor in both of the reactions catalyzed by the enzyme. The KI values for alanosyl-AICOR were approximately 1.5 and 1.3 microM in the SAICAR and adenylosuccinate cleavage reactions respectively. These KI values were essentially the same as the Km values for the two substrates of adenylosuccinate lyase. They compare with an accumulation of 70 microM alanosyl-AICOR in leukemic nodules of mice treated with alanosine [A. K. Tyagi and D. Cooney, Cancer Res. 40, 4390 (1980)]. Thus, inhibition of adenylosuccinate lyase may account for much of the inhibitory effect exerted by alanosyl-AICOR in vivo. We confirmed the previous observation that alanosyl-AICOR is an inhibitor of adenylosuccinate synthetase.