Coordination properties of 3′,5′-cyclic adenosine monophosphate towards copper(II) ions

The metal binding ability of 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) molecule using copper(II) ion, as an example of biologically available divalent metal ion, was investigated by potentiometry, EPR and differential spectroscopy (UV-Vis, CD). One complex with stoichiometry Cu(3′,5′-cAMP)⁺ was found, where Cu(II) ion is bound by N-7 nitrogen of adenine moiety.     

Influence of cyclic 3′, 5′-AMP on glycolysis in human erythrocytes

Small increases in lactate production and in glucose utilization were observed when human red cells were incubated in Tris-Ringer's medium with 5 mM adenosine 3′, 5′-monophosphate (cyclic 3′, 5′-AMP) or its dibutyryl analogue. 5 mM caffeine facilitated this response whereas 5′-AMP, cyclic 2′, 3′-AMP, and guanosine 3′, 5′-AMP had no effect.     

Structural insight into the mechanism of substrate specificity and catalytic activity of an HD-domain phosphohydrolase: the 5'-deoxyribonucleotidase YfbR from Escherichia coli

HD-domain phosphohydrolases have nucleotidase and phosphodiesterase activities and play important roles in the metabolism of nucleotides and in signaling. We present three 2.1-Å-resolution crystal structures (one in the free state and two complexed with natural substrates) of an HD-domain phosphohydrolase, the Escherichia coli 5′-nucleotidase YfbR. The free-state structure of YfbR contains a large cavity accommodating the metal-coordinating HD motif (H33, H68, D69, and D137) and other conserved residues (R18, E72, and D77). Alanine scanning mutagenesis confirms that these residues are important for activity. Two structures of the catalytically inactive mutant E72A complexed with Co²⁺ and either thymidine-5′-monophosphate or 2′-deoxyriboadenosine-5′-monophosphate disclose the novel binding mode of deoxyribonucleotides in the active site. Residue R18 stabilizes the phosphate on the Co²⁺, and residue D77 forms a strong hydrogen bond critical for binding the ribose. The indole side chain of W19 is located close to the 2′-carbon atom of the deoxyribose moiety and is proposed to act as the selectivity switch for deoxyribonucleotide, which is supported by comparison to YfdR, another 5′-nucleotidase in E. coli. The nucleotide bases of both deoxyriboadenosine-5′-monophosphate and thymidine-5′-monophosphate make no specific hydrogen bonds with the protein, explaining the lack of nucleotide base selectivity. The YfbR E72A substrate complex structures also suggest a plausible single-step nucleophilic substitution mechanism. This is the first proposed molecular mechanism for an HD-domain phosphohydrolase based directly on substrate-bound crystal structures.     

Elevation of guanosine 3′,5′-monophosphate level by adenosine in cerebellar slices of guinea pig

Effect of adenosine on the level of guanosine 3′,5′-monophosphate in guinea pig cerebellar slices was investigated. Adenosine increased the concentration of guanosine 3′,5′-monophosphate in the slices 3–4-fold. Upon removal of adenosine from the medium, the concentration of guanosine 3′,5′-monophosphate returned to the initial level. AMP, ADP or ATP also increased the guanosine 3′,5′-monophosphate level to the same extent as adenosine, while adenine or other nucleotides were not effective. In the absence of Ca²⁺ in the incubation medium, adenosine did not increase the concentration of guanosine 3′,5′-monophosphate in cerebellar slices although level of adenosine 3′,5′-monophosphate was elevated by adenosine.Anticholinergic agents, adrenergic blocking agents or antihistaminics did not prevent the increase of guanosine 3′,5′-monophosphate by adenosine indicating that the effect of adenosine was not mediated by the release of neurotransmitters.The combination of adenosine with depolarizing agents showed an additive effect on the level of guanosine 3′,5′-monophosphate indicating that adenosine increased the level of guanosine 3′,5′-monophosphate by a different mechanism from the depolarization.     

A new,sensitive ecto-5′-nucleotidase assay for compound screening

Ecto-5′-nucleotidase (eN) is a membrane-bound enzyme that hydrolyzes extracellular nucleoside-5′-monophosphates yielding the respective nucleoside and phosphate. Increased levels of eN expression have been observed in many cancer cells. By increasing extracellular adenosine concentrations, they contribute to their proliferative, angiogenic, metastatic, and immunosuppressive effects. Therefore, eN is of considerable interest as a novel drug target for the treatment of cancer as well as of inflammatory diseases. In this study, we developed, optimized, and applied a highly sensitive radiometric assay using [³H]adenosine-5′-monophosphate (AMP) as a substrate. The reaction product [³H]adenosine was separated from [³H]AMP by precipitation of the latter with lanthanum chloride and subsequent filtration through glass fiber filters. Conditions were optimized to reproducibly collect the [³H]adenosine-containing filtrate used for quantitative determination. Validation of the assay yielded a mean Z′ factor of 0.73, which demonstrates its suitability for high-throughput screening. The new assay shows a limit of detection that is at least 30-fold lower than those of common colorimetric methods (e.g., optimized malachite green assay and capillary electrophoresis-based assay procedures), and it is also superior to a recently developed luciferase-based assay.     

A bulky platinum triamine complex that reacts faster with guanosine 5′-monophosphate than with N-acetylmethionine

A bulky platinum triamine complex, [Pt(Me₅dien)(NO₃)]NO₃ (Me₅dien = N,N,N′,N′,N′′-pentamethyldiethylenetriamine) has been prepared and reacted in D₂O with N-acetylmethionine (N-AcMet) and guanosine 5′-monophosphate (5′-GMP); the reactions have been studied using ¹H NMR spectroscopy. Reaction with 5′-GMP leads to two rotamers of [Pt(Me₅dien)(5′-GMP-N7)]⁺. Reaction with N-AcMet leads to formation of [Pt(Me₅dien)(N-AcMet-S)]⁺. When a sample with equimolar mixtures of [Pt(Me₅dien)(D₂O)]²⁺, 5′-GMP, and N-AcMet was prepared, [Pt(Me₅dien)(5′-GMP-N7)]⁺ was the dominant product observed throughout the reaction. This selectivity is the opposite of that observed for a similar reaction of [Pt(dien)(D₂O)]²⁺ with 5′-GMP and N-AcMet. To our knowledge, this is the first report of a platinum(II) triamine complex that reacts substantially faster with 5′-GMP than with N-AcMet; the effect is most likely due to steric clashes between the methyl groups of the Me₅dien ligand and the N-AcMet.     

Inhibition of thyroid secretion by sodium fluoride in vitro

NaF mimicked the activation by thyrotropin of iodide binding to proteins and of glucose C-I oxidation but not the accumulation of intracellular colloid droplets or the stimulation of secretion in dog thyroid slices in vitro. On the contrary, NaF inhibited the two latter thyrotropin effects. The inhibitory action of F⁻ was partially relieved by the addition of glucose to the medium; it was mimicked by sodium oxamate. These data suggest that NaF depresses the endocytosis of colloid and thyroid secretion by inhibiting aerobic glycolysis in the follicular cell. NaF inhibited the activation of colloid droplet accumulation and secretion by N⁶,O^2′-dibutyryl-adenosine 3′,5′-monophosphate (dibutyryl cyclic AMP) and the accumulation of cyclic AMP in thyrotropin-stimulated slices. This suggests an inhibition at the level of both cyclic AMP accumulation and cyclic AMP action. The inhibition by NaF and sodium oxamate of colloid droplet formation and thyroid secretion but not of glucose C-I oxidation in stimulated slices further confirms our conclusion that the latter effect is not merely a consequence of the activation by thyrotropin of colloid endocytosis.     

Enzymatic characteristics of two novel Myxococcus xanthus enzymes, PdeA and PdeB, displaying 3′,5′- and 2′,3′-cAMP phosphodiesterase, and phosphatase activities

Myxococcus xanthus PdeA and PdeB, enzymes homologous to class III 3′,5′-cyclic nucleotide phosphodiesterases, hydrolyzed 3′,5′- and 2′,3′-cyclic AMP (cAMP) to adenosine, and also demonstrated phosphatase activity toward nucleoside 5′-tri-, 5′-di-, 5′- and 3′-monophosphates with highest activities for nucleoside 5′-monophosphates. The substrate specificities of PdeA and PdeB show no similarity to that of any known cNMP phosphodiesterase, nucleotidase, or phosphatase. The enzyme activities of PdeA and PdeB were stimulated by 50 μM Mn²⁺ or Co²⁺. The K_m values of PdeA and PdeB for 3′,5′-cAMP, 2′,3′-cAMP, 5′-ATP, and 5′-AMP were in the low micromolar range (1.4-12.5  μM).     

A continuous spectrophotometric assay for monitoring adenosine 5′-monophosphate production

A number of biologically important enzymes release adenosine 5′-monophosphate (AMP) as a product, including aminoacyl–tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5′-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5′-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD⁺) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses.     

2′,3′-cAMP hydrolysis by metal-dependent phosphodiesterases containing DHH, EAL, and HD domains is non-specific: Implications for PDE screening

The recent report of 2′,3′-cAMP isolated from rat kidney is the first proof of its biological existence, which revived interest in this mysterious molecule. 2′,3′-cAMP serves as an extracellular adenosine source, but how it is degraded remains unclear. Here, we report that 2′,3′-cAMP can be hydrolyzed by six phosphodiesterases containing three different families of hydrolytic domains, generating invariably 3′-AMP but not 2′-AMP. The catalytic efficiency (k_cat/K_m) of each enzyme against 2′,3′-cAMP correlates with that against the widely used non-specific substrate bis(p-nitrophenyl)phosphate (bis-pNPP), indicating that 2′,3′-cAMP is a previously unknown non-specific substrate for PDEs. Furthermore, we show that the exclusive formation of 3′-AMP is due to the P-O2′ bond having lower activation energy and is not the result of steric exclusion at enzyme active site. Our analysis provides mechanistic basis to dissect protein function when 2′,3′-cAMP hydrolysis is observed.     

Reactivity of [Ru(pac)(H2O)] (pac = polyaminocarboxylate) complexes with 5′-nucleotides and their antitumor activity

The reaction of [Ru^III(edta)(H₂O)]⁻ (edta⁴⁻ = ethylenediaminetetraacetate) and [Ru^III(hedtra)(H₂O)] (hedtra³⁻ = N-hydroxyethylethylenediaminetriacetate) with various purine based 5′-nucleotides (Nu) viz. adenosin-5′-monophosphate (AMP), guanosin-5′-monophosphate (GMP), inosin-5′-monophosphate (IMP) was studied kinetically as a function of [Nu] at various temperatures (15-35 °C) at a fixed pH (4.5). Kinetic results suggest that the binding of 5′-nucleotides takes place in a rapid [Nu] dependent rate-determining step. Kinetic data and activation parameters are accounted for the operation of an associative mechanism. The antitumor activities of both [Ru^III(edta)(H₂O)]⁻ (1) and [Ru^III(hedtra)(H₂O] (2) have been evaluated using MCF-7 (breast cancer), NCI-H460 (lung cancer) and SF-268 (CNS) cell lines.     

The X-ray crystal structure of APR-B,an atypical adenosine 5′-phosphosulfate reductase from Physcomitrella patens

Sulfonucleotide reductases catalyse the first reductive step of sulfate assimilation. Their substrate specificities generally correlate with the requirement for a [Fe₄S₄] cluster, where adenosine 5′-phosphosulfate (APS) reductases possess a cluster and 3′-phosphoadenosine 5′-phosphosulfate reductases do not. The exception is the APR-B isoform of APS reductase from the moss Physcomitrella patens, which lacks a cluster. The crystal structure of APR-B, the first for a plant sulfonucleotide reductase, is consistent with a preference for APS. Structural conservation with bacterial APS reductase rules out a structural role for the cluster, but supports the contention that it enhances the activity of conventional APS reductases.     

Crystallographic Analysis of the Reaction Cycle of 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase,a Unique Member of the 2H Phosphoesterase Family

2H phosphoesterases catalyze reactions on nucleotide substrates and contain two conserved histidine residues in the active site. Very limited information is currently available on the details of the active site and substrate/product binding during the catalytic cycle of these enzymes. We performed a comprehensive X-ray crystallographic study of mouse 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), a membrane-associated enzyme present at high levels in the tetrapod myelin sheath. We determined crystal structures of the CNPase phosphodiesterase domain complexed with substrate, product, and phosphorothioate analogues. The data provide detailed information on the CNPase reaction mechanism, including substrate binding mode and coordination of the nucleophilic water molecule. Linked to the reaction, an open/close motion of the β5–α7 loop is observed. The role of the N terminus of helix α7—unique for CNPase in the 2H family—during the reaction indicates that 2H phosphoesterases differ in their respective reaction mechanisms despite the conserved catalytic residues. Furthermore, based on small-angle X-ray scattering, we present a model for the full-length enzyme, indicating that the two domains of CNPase form an elongated molecule. Finally, based on our structural data and a comprehensive bioinformatics study, we discuss the conservation of CNPase in various organisms.     

Crystal Structure of the ATPPase Subunit and Its Substrate-Dependent Association with the GATase Subunit: A Novel Regulatory Mechanism for a Two-Subunit-Type GMP Synthetase from Pyrococcus horikoshii OT3

Guanosine 5′-monophosphate synthetase(s) (GMPS) catalyzes the final step of the de novo synthetic pathway of purine nucleotides. GMPS consists of two functional units that are present as domains or subunits: glutamine amidotransferase (GATase) and ATP pyrophosphatase (ATPPase). GATase hydrolyzes glutamine to yield glutamate and ammonia, while ATPPase utilizes ammonia to convert adenyl xanthosine 5′-monophosphate (adenyl-XMP) into guanosine 5′-monophosphate. Here we report the crystal structure of PH-ATPPase (the ATPPase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon Pyrococcus horikoshii OT3). PH-ATPPase consists of two domains (N-domain and C-domain) and exists as a homodimer in the crystal and in solution. The N-domain contains an ATP-binding platform called P-loop, whereas the C-domain contains the xanthosine 5'-monophosphate (XMP)-binding site and also contributes to homodimerization. We have also demonstrated that PH-GATase (the glutamine amidotransferase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon P. horikoshii OT3) alone is inactive, and that all substrates of PH-ATPPase except for ammonia (Mg²⁺, ATP and XMP) are required to stabilize the active complex of PH-ATPPase and PH-GATase subunits.     

Exploring water-soluble Pt(II) complexes of diethylenetriamine derivatives functionalized at the central nitrogen. Synthesis, characterization, and reaction with 5′-GMP

The aims of our program are to develop coordination complexes that can be used as selective probes, fluorescent agents and inorganic medicinal agents. In order to accomplish this, the design, synthesis, characterization and X-ray structure of new water-soluble monofunctional Pt(II) complexes with useful spectroscopic properties for assessing metal binding to biomolecules were investigated. Two diethylenetriamine (dien) derivatives, 2-(bis(2-aminoethyl)amino)acetic acid (acdien) and N′-[7-(acetamido)-4-(trifluoromethyl)coumarin]diethylenetriamine (atfcdien), were used. The latter was designed to allow the fluorophore group, 7-amino-4-(trifluoromethyl)coumarin (atfc), to be attached to metal centers through the dien moiety. ¹H NMR spectroscopy and X-ray crystallography were employed to characterize the [Pt(atfcdien)Br][Pt(Me₂SO)Br₃] (8a) and [Pt(acdien)Br]Br (9a) complexes. ¹H NMR and fluorescence spectroscopic methods were used to characterize the [Pt(atfcdien)Br]Br (8b) and [Pt(acdien)Br]Br (9a) complexes. ¹H NMR studies of the monofunctional [Pt(acdien)Br]Br (9a) complex conducted to examine its interaction with guanosine 5′-monophosphate (5′-GMP) in D₂O solutions revealed one downfield-shifted H8 and one downfield-shifted H1′ signal, consistent with 5′-GMP binding via N7 and fast rotation about the Pt-N7 bond.     

Structural Basis of Substrate Specificity and Selectivity of Murine Cytosolic 5′-Nucleotidase III

Cytosolic 5′-nucleotidase III (cN-III) is responsible for selective degradation of pyrimidine 5′-monoribonucleotides during maturation of reticulocytes to erythrocytes. The lack of this enzymatic activity due to genetic aberrations or lead poisoning results in a mild to moderate nonspherocytic hemolytic anemia. In affected individuals, pyrimidine nucleotides as well as their precursor polymers and their off-path metabolites accumulate in erythrocytes, interfering with their proper function in ways that are not yet fully understood. This report describes the first X-ray structure of a catalytically inactivated variant of murine cN-III with a natural substrate, uridine 5′-monophosphate, in the active site at 1.74 Å resolution. The structure captures in an atomic detail the closed conformation that cN-III adopts upon substrate binding. Structure and sequence analysis coupled with enzymatic characterization of several mutants confirmed that the aromatic ring of a nitrogenous base of substrate nucleotide is stabilized by parallel π-stacking interactions with conserved aromatic rings of Trp113 and His68. The nitrogenous base is further stabilized by T-shaped stacking with the conserved aromatic ring of Tyr114, as well as by polar contacts with side chains of Thr66 and Ser117. Two water molecules help to stabilize the nucleotide binding by bridging it to protein residues Asp72 and His68 via hydrogen bonds. Finally, fully conserved Glu96 is responsible for recognition of ribose ring via two hydrogen bonds. The presented substrate complex structure elucidates how cN-III achieves specificity for pyrimidine 5′-nucleotides and how it selects against purine 5′-nucleotides.     

Platinum(II) triammine antitumour complexes: structure-activity relationship with guanosine 5′-monophosphate (5′-GMP)

The multinuclear (¹H, ¹⁵N, ³¹P and ¹⁹⁵Pt) NMR spectroscopies, ES-MS and HPLC have been employed to investigate the structure-activity relationship for the reactions between guanosine 5′-monophosphate (5′-GMP) and the platinum(II)-triamine complexes of the general formulation cis-[Pt(NH₃)₂(Am)Cl]NO₃ (where Am represents a substituted pyridine). The order of reaction rate of the reactions was found to be: 3-phpy > 4-phpy > py > 4-mepy > 3-mepy > 2-mepy. The two basic factors, steric and electronic, were attributed to the order of the binding rate constants. A possible mechanism of the reaction of cis-[Pt(NH₃)₂(Am)Cl]⁺ with 5′-GMP suggested that the reactions proceed via direct nucleophilic attack and no loss of ammonia. cis-[Pt(NH₃)₂(Am)Cl]⁺ binds to the N7 nitrogen of the guanine residue of 5′-GMP to form a coordinate bond with the Pt metal centre. This mechanism is apparently different from that of cisplatin. The pK_a value of cis-[Pt(NH₃)₂(4-mepy)(H₂O)](NO₃)₂ (5.63) has been determined at 298 K by the use of distortionless enhancement by polarization transfer (DEPT) ¹⁵N NMR spectroscopy and compared to the pK_a value of cis-[PtCl(H₂O)(NH₃)₂]⁺.     

Binding of 2′-Amino-2′-Deoxycytidine-5′-Triphosphate to Norovirus Polymerase Induces Rearrangement of the Active Site

Crystal structures of a genogroup II.4 human norovirus polymerase bound to an RNA primer-template duplex and the substrate analogue 2′-amino-2′-deoxycytidine-5′-triphosphate have been determined to 1.8 Å resolution. The alteration of the substrate-binding site that is required to accommodate the 2′-amino group leads to a rearrangement of the polymerase active site and a disruption of the coordination shells of the active-site metal ions. The mode of binding seen for 2′-amino-2′-deoxycytidine-5′-triphosphate suggests a novel molecular mechanism of inhibition that may be exploited for the design of inhibitors targeting viral RNA polymerases.     

A simultaneous protein-binding assay for adenosine 3':5'-monophosphate in biological materials.

Adenosine 3′:5′-monophosphate (cyclic AMP) and guanosine 3′:5′-monophosphate (cyclic GMP) have been determined simultaneously by combining individual protein binding assays using different isotopically labeled cyclic nucleotides. Preparations of cyclic AMP-binding protein from beef adrenal cortex and cyclic GMP-binding protein from the fat body of silkworm pupae ($\text{Bombyx mori}$) have been used for the assay. The method allows the analysis of cyclic AMP and cyclic GMP levels in crude extracts without any purification. The assay has been applied to hormone-stimulated Mouse liver and phorbol ester-treated Rat embryo cells.