5'-deoxy-5'-thioanalogs of adenosine and inosine 5'-monophosphate: studies with 5'-nucleotidase and alkaline phosphatase

The interaction of 5'-deoxy-5'-thioadenosine 5'-monophosphate (A(S)MP) and 5'-deoxy-5'-thioinosine 5'-monophosphate (I(S)MP) with snake venom, 5'nucleotidase, and calf intestinal mucosa alkaline phosphatase has been characterized. The substrates, A(S)MP and I(S)MP, are analogs of adenosine 5'-monophosphate and inosine 5'-monophosphate in which sulfur replaces oxygen as the bridge between the 5'-carbon of the ribose and the phosphorous. The P-S bond of both A(S)MP and I(S)MP was hydrolyzed by alkaline phosphatase producing the corresponding thionucleoside as a reaction product. The Km for A(S)MP was 270 microM and the V for alkaline phosphatase was 110 nmol/min/mg (8% of the V for AMP), whereas the corresponding values for I(S)MP were 300 microM and 530 nmol/min/mg protein, respectively. In contrast, 5'-nucleotidase did not catalyze hydrolysis of either A(S)MP or I(S)MP. A(S)MP and I(S)MP were competitive inhibitors of the 5'-nucleotidase hydrolysis of AMP and IMP, respectively, with Ki values of 975 and 13 microM. Decreasing the pH of the reaction from 8.1 to 7.1 lowered the Ki for I(S)MP by 100-fold, to a value of 0.15 microM.     

Conformation, hydrogen bonding and aggregate formation of guanosine 5'-monophosphate and guanosine in dimethylsulfoxide.

The tetrabutylammonium salt of guanosine 5'-monophosphate (5'-GMP) dissolves in DMSO-d6 forming aggregated species which exhibit some properties of reverse micelles. 1H NOESY experiments show that the 5'-GMP adopts the syn conformation about the glycosidic bond. Molecular mechanics calculations reveal a stable structure with this conformation in which the phosphate group and the amino group of the base are in close enough proximity to hydrogen bond. In contrast inosine 5'-monophosphate in DMSO-d6, which has no NH2 group for hydrogen bond stabilization of the syn conformation, is shown by NMR to have the anti structure. Guanosine in DMSO-d6 behaves differently from 5'-GMP. Guanosine adopts the anti conformation and forms a symmetric dimer via hydrogen bonding between the N3 and NH2 of the bases.     

A slowly dissociating form of the cell surface cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum.

Experiments using a phosphodiesterase-minus mutant of Dictyostelium discoideum indicate that ligand-induced loss of cell surface cyclic adenosine 3':5'-monophosphate binding sites (down regulation) can be evoked with concentrations of cyclic adenosine 3':5'-monophosphate as low as 10(-8) M. The loss of receptor sites is observed after 5 min of cell preincubation with cyclic adenosine 3':5'-monophosphate and can be as extensive as 75 to 80%. This decrease in binding sites is correlated with the appearance of a slowly dissociating cyclic adenosine 3':5'-monophosphate binding component. Radioactive cyclic adenosine 3':5'-monophosphate bound to this form of receptor cannot be competed for by nonradioactive cyclic adenosine 3':5'-monophosphate or adenosine 5'-monophosphate and is not accessible to hydrolysis by cyclic adenosine 3':5'-monophosphate phosphodiesterase. The extent of appearance of this binding component is dependent upon the concentration of cyclic adenosine 3':5'-monophosphate used to elicit the down regulation response and the temperature of the incubation medium.     

Preparation and properties of N6-monobutyryl adenosine 5'-monophosphate. A major hepatic metabolite of N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate.

The synthesis and properties of N6-monobutyryl adenosine 5'-monophosphate are described. The properties of synthesized monobutyryl nucleotide have been compared to those of a metabolite recognized in previous studies (Castagna, M. C., Palmer, W.K., and Walsh, D.A. (1977) Arch. Biochem. Biophys. 181, 46-60) as the major radioactive product produced in the liver upon perfusion with N6,O2'-dibutyryl cyclic [3H]adenosine 3':5'-monophosphate. By the criteria of cochromatography on DEAE-cellulose and in three thin layer chromatographic systems and from equivalent rates of alkaline hydrolysis, N6-monobutyryl adenosine 5'-monophosphate has been identified as a major hepatic metabolite of N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate.     

F Nuclear Magnetic Resonance Analysis of 5-Fluorouracil Metabolism in Four Differently Pigmented Strains of Nectria haematococca

F nuclear magnetic resonance spectroscopy was used to study the metabolism of 5-fluorouracil in four strains of Nectria haematococca which displayed similar sensitivities to growth inhibition by this compound but differed in their pigmentation. The major metabolites, 5-fluorouridine and alpha-fluoro-beta-alanine, were excreted into the medium by all four strains. The classical ribofluoronucleotides (5-fluorouridine-5'-monophosphate, -diphosphate, and -triphosphate) and alpha-fluoro-beta-alanine were identified in the acid-soluble fraction of perchloric acid extracts of mycelia. Two hydrolysis products of 5-fluorouracil incorporated into RNA were found in the acid-insoluble pool. They were unambiguously assigned to 5-fluorouridine-2'-monophosphate and 3'-monophosphate with specific hydrolysis reactions on isolated RNA. The lack of fluorodeoxyribonucleotides and the fact that the four strains incorporated similar amounts of fluororibonucleotides into their RNAs strongly suggest an RNA-directed mechanism of cytotoxicity for 5-fluorouracil. The heavily pigmented wild type differed from the three low-pigmented strains in its low uptake of 5-fluorouracil and, consequently, in its reduced biosynthesis of 5-fluorouridine and alpha-fluoro-beta-alanine. At present, it is not clear whether this change in 5-fluorouracil metabolism is a side effect of pigment production or results from another event.     

3',5'-Cyclic nucleotide phosphodiesterase activity of the sulphatase A of ox liver

The sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver hydrolyses adenosine 3',5'-monophosphate (cyclic AMP) to adenosine 5'-phosphate at an optimum pH of approx. 4.3, close that for the hydrolysis of cerebroside sulphate, a physiological substrate for sulphatase A. The Km is 11.6 mM for cyclic AMP. On polyacrylamide gel electrophoresis sulphatase A migrates as a single protein band which coincides with both the arylsulphatase and phosphodiesterase activities, suggesting that these are due to a single protein. Cyclic AMP competitively inhibits the arylsulphatase activity of sulphatase A, showing that both activities are associated with a single active site on the enzyme. sulphatase A also hydrolyses guanosine 3',5'-monophosphate, but not uridine 3',5'-monophosphate nor adenosine 2',3'-monophosphate.     

Soluble NTPDase: An additional system of nucleotide hydrolysis in rat blood serum

The participation of a nucleoside triphosphate diphosphohydrolase in the nucleotide hydrolysis by rat blood serum was evaluated. Nucleoside triphosphate diphosphohydrolase and phosphodiesterase are enzymes possibly involved in ATP and ADP hydrolysis. The specific activity of the phosphodiesterase activity (using thymidine 5'-monophosphate p-nitrophenyl ester as substrate) was 4.92 +/- 0.73 (mean +/- SD, n = 10) nmol p-nitrophenol.min(-1).mg(-1) protein and the specific activities for ATP and ADP were 1.31 +/- 0.37 (mean +/- SD, n = 7) and 1.36 +/- 0.25 (mean +/- SD, n = 5) nmol Pi.min(-1).mg(-1) protein, respectively. A competition plot demonstrated that ATP and ADP hydrolysis occurs at the same active site. The effect of suramin and phenylalanine on ATP, ADP and thymidine 5'-monophosphate p-nitrophenyl ester hydrolysis was investigated. The results were opposite considering the hydrolysis of ATP and ADP and that of the substrate marker for the enzyme phosphodiesterase. These results are indicative of the presence of, at least, two enzymes participating in the serum nucleotide hydrolysis. The presence of cAMP did not affect the hydrolysis velocity of ATP and ADP, while thymidine 5'-monophosphate p-nitrophenyl ester hydrolysis was inhibited by cAMP by approximately 47%, suggesting that the hydrolysis of the ATP and ADP, under our assay conditions, occurs at a different site from the phosphodiesterase site. Both enzyme activities, in the rat blood serum, may be involved in the modulation of the nucleotide/nucleoside ratio in the circulation, serving an in vivo homeostatic and antithrombotic function. In addition, the phosphodiesterase may act on DNA or RNA liberated upon tissue injury and/or cell death.     

Effects of 8-substituted adenosine 3',5'-monophosphate derivatives on high Km phosphodiesterase activity.

Most of twenty-one 8-substitued adenosine 3',5'-monophosphate derivatives were found to inhibit competitively the hydrolysis of adenosine 3'5'-monophosphate by partially purified high Km (Michaelis-Menten constant) phosphodiesterase from hog brain cortex, which had one active site at high concentration of adenosine 3',5'-monophosphate (0.3 to 4.0 mM). The Ki value for the 8-substituted alkylaminoadenosine 3'5'-monophosphate derivative was found to decrease with increasing unbranched carbon chain of the substituent, and a minimum value was obtained in the case of 8-octylaminoadenosine 3',5'-monophosphate. The Ki value, however, increased gradually as the substituent of derivative became longer than that of 8-octylminoadenosine 3'5'-monophosphate. The similar phenomenon was observed in the 8-substituted alkylthioadenosine 3',5'-monophosphate. The standard affinity for adenosine 3,5'-monophosphate of the high Km phosphodiesterase was 5.0 kcal/mol, which was calculated from Km. The standard affinity for 8-hexylthioadenosine 3',5'-monophosphate, which inhibited most strongly the enzyme activity, was 7.2 kcal/mol. The difference (2.2 kcal/736) between the standard affinity for adenosine 3',5'-monphosphate and that for 8-hexylthioadenosine 3',5'-monophosphate seems to be based on the partial affinity for the substituent (hexylthio group) of the active site on the enzyme or its neighborhood. A characteristic similar interrelation between substituent length of derivatives and their inhibitory effect on the enzyme activity was observed similarly in two different series of derivatives, 8-substituted alkylaminoadenosine 3',5'-monophosphate and alkylthioadenosine 3',5'-monophosphate. The results may indicate the characteristic structure of the active site of the enzyme or its neighborhood.     

The mechanisms of action of cAMP. A quantum chemical study

Quantum chemical calculations were performed on the formation of intermediates with trigonal bipyramidal (TBP) configurations in the hydrolysis of adenosine 3',5'-monophosphate (cAMP) with phosphodiesterases and the activation of protein kinases by cAMP. The results show that in the reaction sequence concerning the hydrolysis of cAMP with phosphodiesterase the TBP intermediate must possess an equatorial-apical cyclic phosphate ring with the 3'-oxygen atom in the apical position. This could be an additional reason for the sensitivity of the 3' position in cAMP towards modifications in comparison with the 5' position. According to the calculations, a mechanistic model is presented for the enzymatic hydrolysis of cAMP with the involvement of a covalently bonded enzyme-nucleotide intermediate. Also a model is offered for the activation of protein kinase by cAMP. The activation of protein kinase is assumed to proceed via diequatorial-ring-positioned TBP intermediates resulting in the formation of a covalent bond between cAMP and the protein kinase with retention of the cyclic phosphate ring. It seems likely that the enzyme-nucleotide intermediate enforces a conformational change in the enzyme, which causes the dissociation of the regulatory and catalytic subunit of the protein kinase, necessary for a physiological response.     

Molecular orbital studies on the conformations of 8-amino- and 8-dimethylaminoadenosine 5'-monophosphate

The quantum mechanical PCILO method has been applied for the determination of conformational properties of 8-amino- and 8-dimethylaminoadenosine 5'-monophosphate. Contrary to other 8-substituted nucleotides the amino derivative shows a preference for an anti arrangement about the glycosidic bond. This conformation is stabilized by an intramolecular hydrogen bond between the purine and the exocyclic group. 8-dimethylamino-adenosine-5'-monophosphate adopts the syn conformation with slightly rotated dimethylamino group. There is, however, a local minimum for the anti form associated with the unusual value of chiCN = 300 degrees. This minimum is probably populated when the nucleotide is bound to lactate dehydrogenase apoenzyme. No particularly strong interactions are necessary for the stabilization of the anti form. The computations account satisfactorily for the available experimental data.     

Methylation of desmethyl analogue of Y nucleosides. Wyosine from guanosine.

Wyosine la, one of the fluorescent hypermodified Y nucleosides found in tRNAsPhe, was synthesized chemically from its biogenetic precursor guanosine 2. The route involved transformation of 2 into the tricyclic structure 3a and subsequent methylation at N-4. The major products of various methylation procedures were isomers of wyosine, methylated at N-5 (3b) or at N-1 (4). Mesoionic compound 4 is a new analogue of 7-methylguanosine 5, modified nucleoside occurring in the unique positions in transfer, messenger and ribosomal RNAs. The chromatographic and spectral characteristics of wyosine and its isomers is given.     

2-Methylwyosine,a Nucleoside With Restricted Anti Conformation in the East Region Enforced by Nucleobase Moiety Modification: Synthesis and Conformational Analysis by NMR and Molecular Dynamics

GRAPHICAL ABSTRACT

We synthesized a new 2-methyl derivative of wyosine using a multistep procedure starting from guanosine. We examined different synthetic paths and optimized the conditions for each step. Based on MD calculations and analysis of the ³ J _HH and J _C1′H1′ of the ribose moiety, we discovered that the sugar part adopted conformation specific for the East region rarely occurring in solution. This unusual conformational preference is probably due to steric repulsions between the methyl group at position 2 and the 5′-CH₂OH group. We observed that N-glycosidic bond stability weakened 14-fold upon the introduction of the methyl group in position 2 compared with wyosine.     

Determination of the Tautomeric Populations of the Y-Base

Abstract

Determination of tautomeric population of the Y-base in 3 by ¹³C-NMR spectroscopy has shown that its most thermodynamically preferred tautomeric form is the N¹-H tautomer (～ 95 %) which explains its chemical reactivity in glycosylation reaction and also in the facile, lewis acid promoted isomerization of wyosine-2′,3′,5-O-triacetate 5 to its N¹-isomer 4. It is likely that the consequence of occurrence of natural wyosine in the thermodynamically unfavoured N³-form is its unusual lability across the glycosyl bond under mild acidic conditions.     

Low nanogram detection of nucleotides using fast atom bombardment-mass spectrometry

The effect of trimethylsilyl (TMS) derivatization on detection limits of mononucleotides in fast atom bombardment-mass spectrometry (FAB-MS) was examined. FAB-MS methods were developed to optimize sensitivity using adenosine 5'-monophosphate as a model compound and then applied to reference standards of two clinically important nucleotides: tricyclic nucleoside-5'-monophosphate (TCNMP) and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP). The detection limit for the TMS derivative of TCNMP was 2.5-5 ng/microliters and less than 2.5 ng/microliters for FdUMP as its TMS derivative. This is greater than two orders of magnitude more sensitive than the FAB-MS analysis of the corresponding free compounds. These low detection limits for the TMS derivatives were obtained using a narrow scan range, signal averaging, detection in the negative ion mode, and 3-nitrobenzyl alcohol as the matrix. Hydrolysis of one or more of the labile TMS groups did occur, with the extent of hydrolysis being greatest in the more protic matrices.     

Transient kinetics of a cGMP-dependent cGMP-specific phosphodiesterase from Dictyostelium discoideum

Chemotactic stimulation of Dictyostelium discoideum cells induces a fast transient increase of cGMP levels which reach a peak at 10 s. Prestimulation levels are recovered in approximately 30 s, which is achieved mainly by the action of a guanosine 3',5'-monophosphate cGMP-specific phosphodiesterase. This enzyme is activated about fourfold by low cGMP concentrations. The phosphodiesterase has two distinct cGMP-binding sites: a catalytic site and an activator site. cAMP does not bind to either site; inosine 3',5'-monophosphate (cIMP) binds only to the catalytic site, whereas 8-bromoguanosine 3',5'-monophosphate (c-b8-GMP) preferentially binds to the activator site. For detailed kinetical measurements we have used [3H]cIMP as the substrate and c-b8-GMP as the activator. c-b8-GMP activated the hydrolysis of [3H]cIMP by reducing the Km, whereas the Vmax was not altered. The hydrolysis of [3H]cIMP was measured at 5-s intervals by using a new method for the separation of 5'-nucleotides from cyclic nucleotides. The hydrolysis of [3H]cIMP by nonactivated enzyme or by preactivated enzyme was linear with time, which indicates that a steady state is reached at the catalytic site within 5 s after addition of the substrate. In contrast, the hydrolysis of [3H]cIMP immediately after activation by 0.1 microM c-b8-GMP was not linear with time, but increased in a quasi-exponential manner with a time constant of 21 s. This suggests that a steady state at the activator site is only reached in 30-45 s after addition of the activator. The on-rate of activation (k1) was 3 X 10(5) M-1s-1 for c-b8-GMP and 1.4 X 10(5) M-1s-1 for cGMP. The off-rate of activation (k-1) was 0.03 s-1 for both c-b8-GMP and cGMP. The significance of these kinetic constants for the chemoattractant-mediated cGMP response in vivo is discussed.     

Structural basis for the decarboxylation of orotidine 5'-monophosphate (OMP) by Plasmodium falciparum OMP decarboxylase

Orotidine 5'-monophoshate decarboxylase (OMPDC) catalyses the decarboxylation of orotidine 5'-monophosphate (OMP) to uridine 5'-monophosphate (UMP). Here, we report the X-ray analysis of apo, substrate or product-complex forms of OMPDC from Plasmodium falciparum (PfOMPDC) at 2.7, 2.65 and 2.65 A, respectively. The structural analysis provides the substrate recognition mechanism with dynamic structural changes, as well as the rearrangement of the hydrogen bond array at the active site. The structural basis of substrate or product binding to PfOMPDC will help to uncover the decarboxylation mechanism and facilitate structure-based optimization of antimalarial drugs.     

Determination of the mechanism of orotidine 5'-monophosphate decarboxylase by isotope effects

Orotidine 5'-monophosphate shows a (15)N isotope effect of 1.0036 at N-1 for decarboxylation catalyzed by orotidine 5'-monophosphate decarboxylase. Picolinic acid shows a (15)N isotope effect of 0.9955 for decarboxylation in ethylene glycol at 190 degrees C, while N-methyl picolinic acid shows a (15)N isotope effect of 1.0053 at 120 degrees C. The transition state for enzymatic decarboxylation of orotidine 5'-monophosphate resembles the transition state for N-methyl picolinic acid in that no bond order changes take place at N-1. This rules out enolization to give a quaternary nitrogen at N-1 in the enzymatic mechanism and suggests a carbanion intermediate stabilized by simple electrostatic interaction with Lys-93. The driving force for the reaction appears to be ground-state destabilization resulting from charge repulsion between the carboxyl of the substrate and Asp-91.     

Hydrolysis of oligoribonucleotides: influence of sequence and length.

The chemical stability of phosphodiester bonds of some oligoribonucleotides in the presence of a cofactor like polyvinylpyrolidine (PVP) is sequence dependent. It was found that pyrimidine-A (YA) and pyrimidine-C (YC) are especially susceptible to hydrolysis. The hydrolyzability of this same phosphodiester bond is dependent on its position in the oligomer. The presence of 3' and 5'-adjacent nucleotides enhances hydrolysis of the UA phosphodiester bond. The acceleration of the hydrolysis of UA by a 5'-adjacent nucleotide is not base dependent. However, a 3'-adjacent purine increases hydrolysis of a UA phosphodiester bond more than a 3'-pyrimidine. The presence of the exoamino group on the 3'-side base (on 6 and 4 position for adenosine and cytidine, respectively) of YA or YZ phosphodiester bond is required for hydrolysis.     

The action of a water-soluble carbodiimide on adenosine-5''-polyphosphates.

When aqueous solutions of adenosine-5'-mono-, di-, or triphosphates are treated with a water soluble carbodiimide the major product is the expected diadenosine-5'-5'-polyphosphate. The yields of these pyrophosphates are greatly increased in the presence of the Mg2+ ion. Adenosine-5'-tetraphosphate behaves differently. The major product is adenosine-5'-monophosphate. We believe that this hydrolysis occurs via a cyclic trimetaphosphate intermediate.