Binding modes of inhibitors to ribonuclease T1 as studied by nuclear magnetic resonance

The binding modes of inhibitors to ribonuclease T1 (RNase T1) were studied by the analyses of 270-MHz proton NMR spectra. The chemical shift changes upon binding of phosphate, guanosine, 2'-GMP, 3'-GMP, 5'-GMP, and guanosine 3',5'-bis(phosphate) were observed as high field shifted methyl proton resonances of RNase T1. One methyl resonance was shifted upon binding of phosphate and guanosine nucleotides but not upon binding of guanosine. Four other methyl resonances were shifted upon binding of guanosine and guanosine nucleotides but not upon binding of phosphate. From the analyses of nuclear Overhauser effects for the pair of H8 and H1' protons, together with the vicinal coupling constants for the pair of H1' and H2' protons, the conformation of the guanosine moiety as bound to RNase T1 is found to be C3'-endo-syn for 2'-GMP and 3'-GMP and C3'-endo-anti for 5'-GMP and guanosine 3',5'-bis(phosphate). These observations suggest that RNase T1 probably has specific binding sites for the guanine base and 3'-phosphate group (P1 site) but not for the 5'-phosphate group (PO site) or the ribose ring. The weak binding of guanosine 3',5'-bis(phosphate) and 5'-GMP to RNase T1 is achieved by taking the anti form about the glycosyl bond. The productive binding to RNase T1 probably requires the syn form of the guanosine moiety of RNA substrates.     

A 31P-NMR study of the interaction of Mg2+ ions with nucleoside diphosphates.

The interaction of Mg2+ with nucleoside disphosphates : ADP, GDP, CDP and UDP has been studied by phosphorus magnetic resonance spectroscopy in aqueous solution. The results show that these four nucleotides behave similarly, the Mg2+ ion binds to the alpha but not to the beta phosphate moiety. The strength of the interaction of Mg2+ ions with nucleoside diphosphates is weaker than with nucleoside triphosphates. The association of Mg2+ on the phosphate chain is stronger in a neutral than in an acid medium.     

Enzyme specificity: base recognition and hydrolysis of RNA by ribonuclease A

The substrate specificity of pancreatic ribonuclease A is discussed in light of observations based on accurate X-ray structure analysis of several enzyme-nucleotide complexes. A hypothesis for protein-nucleic acid recognition is presented which proposes that: (a) pyrimidine bases in RNA are recognised by ribonuclease due to the charge complementarity of two groups (the amide nitrogen and the side chain oxygen (OG) of threonine 45) of the protein and relevant atoms in the heterocyclic base (O2 and N3 in pyrimidine nucleotides); (b) interaction of the protein with the ribose moiety of the nucleotides is non-specific; and (c) conformational flexibility in the region of the scissile P-O bond is provided by different locations of the phosphoryl oxygens, rather than by an overall translation of the phosphate moiety.     

Alkylation of DNA by melphalan: investigation of capillary liquid chromatography-electrospray ionization tandem mass spectrometry in the study of the adducts at the nucleoside level

Nitrogen mustards are among the oldest cancer chemotherapeutic agents and remain the drugs of choice for treatment of many human cancers. A serious complication of treatment with nitrogen mustards is the increased risk of a secondary leukaemia in long-term survivors because not all alkylating agent interactions with DNA result in cell death. In an earlier study 2'-deoxy-5'-mononucleotide/melphalan adducts have been analysed by us by LC-ES MSMS. In this work we want to present the first results of the analysis of the corresponding 2'-deoxynucleoside/melphalan adducts from DNA hydrolysates by column switching/capillary LC-ES tandem mass spectrometry. Nucleosides, compared to nucleotides, give better chromatographic results and show a good sensitivity under electrospray (+) [ES(+)] ionisation. Several adducts were identified under ES(+) conditions. Mono-alkylated nucleoside adducts alkylated at the base moiety were identified for dGuo, dCyd and dAdo. Structures were identified by recording the low-energy CAD product ion scans. Also a mono-alkylated nucleotide pdA with alkylation position at the phosphate moiety could be detected. This proves that in the case of phosphate alkylation the enzymatic dephosphorylation reaction was inhibited. A Jurkat cell suspension was treated with melphalan (1 mM) and incubated at 37 degrees C (5% CO(2)). After 6 and 48 h, the DNA was isolated and enzymatically hydrolysed. The corresponding nucleoside pool was evaluated with the developed LC-MS method. In the 48-h experiment, one adduct could be identified as a N-7 alkylated dGuo. In the 6-h experiment, no adducts could be found. Additional experiments were done wherein Jurkat-DNA, isolated from a non-treated cell culture, was treated with melphalan. These results were analogous with the data found in melphalan-treated calf thymus DNA. Additionally, we tried to determine the exact alkylation position by interpreting high-resolution fragmentation spectra.     

The catalytic mechanism of indole-3-glycerol phosphate synthase: crystal structures of complexes of the enzyme from Sulfolobus solfataricus with substrate analogue,substrate, and product

Indoleglycerol phosphate synthase catalyzes the ring closure of an N-alkylated anthranilate to a 3-alkyl indole derivative, a reaction requiring Lewis acid catalysis in vitro. Here, we investigated the enzymatic reaction mechanism through X-ray crystallography of complexes of the hyperthermostable enzyme from Sulfolobus solfataricus with the substrate 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate, a substrate analogue and the product indole-3-glycerol phosphate. The substrate and the substrate analogue are bound to the active site in a similar, extended conformation between the previously identified phosphate binding site and a hydrophobic pocket for the anthranilate moiety. This binding mode is unproductive, because the carbon atoms that are to be joined are too far apart. The indole ring of the bound product resides in a second hydrophobic pocket adjacent to that of the anthranilate moiety of the substrate. Although the hydrophobic moiety of the substrate moves during catalysis from one hydrophobic pocket to the other, the triosephosphate moiety remains rigidly bound to the same set of hydrogen-bonding residues. Simultaneously, the catalytically important residues Lys53, Lys110 and Glu159 maintain favourable distances to the atoms of the ligand undergoing covalent changes. On the basis of these data, the structures of two putative catalytic intermediates were modelled into the active site. This new structural information and the modelling studies provide further insight into the mechanism of enzyme-catalyzed indole synthesis. The charged epsilon-amino group of Lys110 is the general acid, and the carboxylate group of Glu159 is the general base. Lys53 guides the substrate undergoing conformational transitions during catalysis, by forming a salt-bridge to the carboxylate group of its anthranilate moiety.     

Tightly bound nucleotides affect phosphate binding to mitochondrial F1-ATPase

The interaction of inorganic phosphate with native and nucleotide-depleted F1-ATPase was studied. F1-ATPase depleted of tightly bound nucleotides loses the ability to bind inorganic phosphate. The addition of ATP, ADP, GTP and GDP but not AMP, restores the phosphate binding. The nucleotides affecting the phosphate binding to F1-ATPase are located at the catalytic (exchangeable) site of the enzyme. The phosphate is thought to bind to the same catalytic site where the nucleotide is already bound. It is thought that ADP is the first substrate to bind to F1-ATPase in the ATP synthesis reaction.     

Interaction of metal ions with nucleic acids. Interaction of copper(II) with pyrimidine nucleosides and their derivatives.

1. In aqueous and non-aqueous solutions, copper(II) interacts with the N-3 of cytidine but not with the carbonyl group oxygens of pyrimidine nucleosides. 2. In aqueous solution, copper(II) interacts with the phosphate group and ribose of pyrimidine nucleotides, and additionally with N-3 of 5'-CMP. 3. Broadening of resonance signals of the H-5 proton of 5'-UMP and C-5 of 5'-UMP and 5'-TMP results probably from the interaction between metal ion and the phosphate group situated in direct vicinity of the above atoms. 4. In the copper(II)-pyrimidine nucleotide complexes in solid state, copper is coordinated with the phosphate group, and in 5'-CMP additionally with the pyrimidine moiety of the nucleotide.     

Stereochemistry of the acyl dihydroxyacetone phosphate acyl exchange reaction

The fatty acid of acyl dihydroxyacetone phosphate can be exchanged enzymatically for another fatty acid. It has been shown that this reaction proceeds by cleavage of the oxygen bound to C-1 of the dihydroxyacetone phosphate (DHAP) moiety rather than by the more common cleavage at the acyl to oxygen bond. In the present study, the stereochemistry of this reaction was defined further; using deuterated substrates and fast atom bombardment-mass spectrometry, it was shown that the fatty acid exchange involves the stereospecific labilization of the pro-R hydrogen at C-1 of the DHAP moiety of acyl DHAP. The mechanism of ether bond formation, in which acyl DHAP is converted to O-alkyl DHAP, also proceeds via labilization of the pro-R hydrogen and cleavage of the fatty acid at the C-1 to oxygen bond. In addition, other workers have provided evidence that the enzyme responsible for the exchange reaction is O-alkyl DHAP synthetase. Therefore, the present results support the hypothesis that the acyl exchange is the reverse reaction of the first step in O-alkyl DHAP synthesis; in both of these reactions the pro-R hydrogen of C-1 of the DHAP moiety of acyl DHAP and the fatty acid moiety are labilized with cleavage of the fatty acid at the DHAP C-1 to oxygen bond.     

A facilitated electron transfer of copper--zinc superoxide dismutase (SOD) based on a cysteine-bridged SOD electrode

The direct electrochemical redox reaction of bovine erythrocyte copper--zinc superoxide dismutase (Cu(2)Zn(2)SOD) was clearly observed at a gold electrode modified with a self-assembled monolayer (SAM) of cysteine in phosphate buffer solution containing SOD, although its reaction could not be observed at the bare electrode. In this case, SOD was found to be stably confined on the SAM of cysteine and the redox response could be observed even when the cysteine-SAM electrode used in the SOD solution was transferred to the pure electrolyte solution containing no SOD, suggesting the permanent binding of SOD via the SAM of cysteine on the electrode surface. The electrode reaction of the SOD confined on the cysteine-SAM electrode was found to be quasi-reversible with the formal potential of 65 +/- 3 mV vs. Ag/AgCl and its kinetic parameters were estimated: the electron transfer rate constant k(s) is 1.2 +/- 0.2 s(-1) and the anodic (alpha(a)) and cathodic (alpha(c)) transfer coefficients are 0.39 +/- 0.02 and 0.61 +/- 0.02, respectively. The assignment of the redox peak of SOD at the cysteine-SAM modified electrode could be sufficiently carried out using the native SOD (Cu(2)Zn(2)SOD), its Cu- or Zn-free derivatives (E(2)Zn(2)SOD and Cu(2)E(2)SOD, E designates an empty site) and the SOD reconstituted from E(2)Zn(2)SOD and Cu(2+). The Cu complex moiety, the active site for the enzymatic dismutation of the superoxide ion, was characterized to be also the electroactive site of SOD. In addition, we found that the SOD confined on the electrode can be expected to possess its inherent enzymatic activity for dismutation of the superoxide ion.     

Nucleotides and inorganic phosphates as potential antioxidants

Highly reactive OH radicals, formed in an iron-ion catalyzed Fenton reaction, are implicated in many pathological conditions. The quest for Fenton reaction inhibitors, either radical scavenger or metal-ion chelator antioxidants, spans the previous decades. Purine nucleotides were previously studied as natural modulators of the Fenton reaction; however, the modulatory role of purine nucleotides remained in dispute. Here, we have resolved this long-standing dispute and demonstrated a concentration-dependent biphasic modulation of the Fenton reaction by nucleotides. By electron spin resonance measurements with 0.1 mM Fe(II), we observed an increase of •OH production at low purine nucleotide concentrations (up to 0.15 mM), while at higher nucleotide concentrations, an exponential decay of •OH concentration was observed. We found that the phosphate moiety, not the nucleoside, determines the pro/antioxidant properties of a nucleotide, suggesting a chelation-based modulation. Furthermore, the biphasic modulation mode is probably due to diverse nucleotide–Fe(II) complexes formed in a concentration-dependent manner. At ATP concentrations much greater than Fe(II) concentrations, multiligand chelates are formed which inhibit the Fenton reaction owing to a full Fe(II) coordination sphere. In addition to natural nucleotides, we investigated a series of base- or phosphate-modified nucleotides, dinucleotides, and inorganic phosphates, as potential biocompatible antioxidants. Ap₅A, inorganic thiophosphate and ATP-γ-S proved highly potent antioxidants with IC₅₀ values of 40, 30, and 10 μM, respectively. ATP-γ-S proved 100 and 20 times more active than ATP and the potent antioxidant Trolox, respectively. In the presence of 30 μM ATP-γ-S no •OH was detected after 5 min in the Fenton reaction mixture. The most potent antioxidants identified inhibit the Fenton reaction by forming full coordination sphere chelates.Patent pending.     

Properties of allosteric nicotinamide mononucleotide glycohydrolase from Azotobacter vinelandii: activation and inhibition

In order to clarify the regulation mechanism of NMN glycohydrolase, a number of purine and pyrimidine nucleotides were tested as activators for the enzyme. Among naturally occurring nucleotides, pppGpp was shown to be the most potent activator (KA = 0.0087 mM). The effectiveness of these nucleotides estimated from Vmax/KA was in the order: pppGpp greater than ppGpp greater than pppppG greater than ppppG greater than pGpp greater than GTP greater than or equal to 2'-GMP greater than or equal to GppppG greater than dGTP greater than ITP greater than GDP = 2'-O-methylGTP. XTP, UTP, dTTP gave no effect. In contrast, ATP, CTP, and 7-methylGTP were inhibitory. A comparison of the data indicated that activation of the enzyme is specific for the base structure, guanine, and that effectiveness of nucleotides as activators results from the negatively charged phosphate moiety. Consistent with these results, inorganic polyphosphates (tri-, tetra-, and pentaphosphate) and 5-phosphoribose 1-pyrophosphate were shown to function as inhibitors of the enzyme. The inhibition mechanism was shown to be competitive with GTP. However, apparent Ki values for these inhibitors were dependent on the activator concentration. Furthermore, a shift of cooperativity type and progressive increase of positive cooperativity concomitant with increase of PRPP concentration were observed. These results indicate that this enzyme may be allosteric and an important regulatory component of pyridine nucleotide cycle metabolism.     

Irreversible inhibition of aldolase by a phosphorylated alpha-dicarbonyl compound

The preparation of a phosphorylated alpha-dicarbonyl compound designed to specifically react with arginine residues of enzymes accepting phosphorylated compounds as effectors is reported, and shown to inhibit rabbit muscle aldolase in a time-dependent and irreversible manner. This irreversible inhibition occured in a buffer devoid of borate ions, suggesting that the presence of the phosphate moiety contributes in the stabilization of the adduct formed with arginine residues. Under the same conditions, the metalloenzyme iron superoxide dismutase, in which an arginine is known to be critical for the catalytic function, is not significantly inhibited.     

Purification and properties of deoxyadenosine kinase from rat liver mitochondria. II. Kinetics and inhibition studies

Kinetic and inhibition studies of partially purified deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76, AdR kinase) from rat liver mitochondria were performed, including reaction properties, specificity of phosphate donors, phosphate acceptors, influence of nucleotides, nucleosides and their derivatives. The results were compared with those obtained from partially purified AdR kinase from rat liver cytosol.     

The creatine kinase system in rat thymus and thymocytes

The content of creatine phosphate, creatine and creatine kinase activity in thymus is shown to be 17.6, 5 and 4 times respectively higher, than in thymocytes isolated from this organ, both the level of adenine nucleotides and adenylate energy charge being practically the same. The creatine phosphate content in thymocytes decreases with addition of papaverine and remains unchanged under the influence of adenosine and concanavalin A. The creatine kinase activity increases considerably during the concanavalin A-induced thymocyte blasttransformation reaction. Creatine inhibits blasttransformation of thymocytes stimulated by this mitogen.     

Inosine nucleosidase from Azotobacter vinelandii

An enzyme catalyzing the hydrolysis of purine nucleosides was found to occur in the extract of Azotobacter vinelandii, strain 0, and was highly purified by ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxylapatite chromatography and gel filtration on Sephadex G-150. A strict substrate specificity of the purified enzyme was shown with respect to the base components. The enzyme specifically attacked the nucleosides without amino groups in the purine moiety: inosine gave the maximum rate of hydrolysis and xanthosine was hydrolyzed to a lesser extent. The pH optimum of inosine hydrolysis was observed from pH 7 to 9, while xanthosine was hydrolyzed maximally at pH 7. The K _m values of the enzyme for inosine were 0.65 and 0.85 mM at pH 7.1 and 9.0, respectively, and the value for xanthosine was 1.2 mM at pH 7.1.Several nucleotides inhibited the enzyme: the phosphate portions of the nucleotides were suggested to be responsible for the inhibition by nucleotides. Although the inhibition of the enzyme by nucleotides was apparently non-competitive type with respect to inosine, allosteric (cooperative) binding of the substrate was suggested in the presence of the inhibitor. The physiological significance of the enzyme was discussed in connection with the degradation and salvage pathways of purine nucleotides.     

Reaction rate constants of superoxide scavenging by plant antioxidants

Plant phenols may exert protective effects by scavenging superoxide, which is implicated in tissue damage and accelerated inactivation of vasorelaxing nitric oxide. Preventing the interaction of superoxide with tissue biomolecules depends not only on the extent of superoxide scavenging but also on scavenging velocity. However, information on superoxide scavenging kinetics of plant phenols is scarce. We describe an improved lucigenin-based chemiluminescence assay for kinetic analysis. The use of potassium superoxide (KO₂) as a nonenzymatic superoxide source allowed simple and reliable determination of the second-order reaction rate constants between superoxide and plant antioxidants at physiologically relevant conditions, avoiding unspecific effects of other reactive oxygen species or superoxide-generating enzymes. We calculated the rate constants for phenols of different structures, ranging from 2.9 × 10³ mol⁻¹ l s⁻¹ for morin to 2.9 × 10⁷ mol⁻¹ l s⁻¹ for proanthocyanidins. Compounds with pyrogallol or catechol moieties were revealed as the most rapid superoxide scavengers, and the gallate moiety was found to be the minimal essential structure for maximal reaction rate constants with superoxide.     

A novel human phosphotransferase highly specific for adenosine.

A novel nucleoside phosphotransferase, referred to as adenosine phosphotransferase (Ado Ptase), was partially purified 1230-fold from human placenta. This enzyme differed from other known nucleoside phosphotransferases in its substrate specificity. Using AMP as the phosphate donor, it readily phosphorylated Ado. Changes in the sugar moiety were tolerated. dAdo and ddAdo were phosphate acceptors and dAMP was a donor. No other nucleotide or nucleoside common in nature displayed appreciable activity as donor or acceptor substrate, respectively. In the absence of nucleoside, the enzyme catalyzed the hydrolysis of AMP, typical of other nucleoside phosphotransferases. However, in the presence of Ado, little, if any, hydrolysis occurred. Ado Ptase had an absolute requirement for a metal cation, with Mg2+ and, to a lesser extent, Mn2+ fulfilling this requisite. The apparent Km for Ado was 0.2 mM. However, the donor AMP displayed cooperativity in both transfer and hydrolytic reactions. This cooperativity was eliminated by nucleotides, 2,3-diphosphoglycerate, and inorganic phosphate. ADP and 2,3-diphosphoglycerate were especially potent. In the presence of these effectors, the apparent Km for AMP was 3.0 mM in the transfer reaction and 4.0 mM in the hydrolytic reaction. Kinetic data suggest that there are two nucleotide binding sites on Ado Ptase, one for the donor, the other for an effector. AMP appeared to bind to both sites. Although this novel enzyme might play a role in the anabolism of nucleoside analogues, the normal physiological role of this nucleoside phosphotransferase is not understood.     

Further characterization of a protein kinase-substrate complex precipitable with Ca2+ from the cytosol fraction of AH-66 hepatoma cells

The properties of a protein kinase-substrate complex precipitated with Ca2+ from the cytosol of AH-66 hepatoma cells were characterized. The endogenous phosphorylation reaction of the complex was little affected by addition of histone, cyclic nucleotides, Ca2+-calmodulin, or Ca2+-phospholipid but was increased about two-fold by addition of casein. The complex contained several phosphate acceptor proteins with molecular weights ranging from 74,000 to 13,000 as analyzed by two-dimensional gel electrophoresis. These phosphate acceptor proteins were specifically concentrated in the complex. The protein kinase in the complex was purified by successive chromatography and proved to be casein kinase 2.     

Nucleotide pool in pho regulon mutants and alkaline phosphatase synthesis in Escherichia coli.

The intracellular nucleotide pool of Escherichia coli W3110 reproducibly changes from conditions of growth in phosphate excess to phosphate starvation, with at least two nucleotides appearing under starvation conditions and two nucleotides appearing only under excess phosphate conditions. Strains bearing a deletion of the phoA gene show the same pattern, indicating that dephosphorylation by alkaline phosphatase is not responsible for the changes. Strains with mutations in the phoU gene, which result in constitutive expression of the pho regulon, show the nucleotide pattern of phosphate-starved cells even during phosphate excess growth. These changes in nucleotides are therefore due to phoU mutation but not to alkaline phosphatase constitutivity. In fact, a phoR (phoR68) mutant strain has the patterns of the wild type in spite of being constitutive for alkaline phosphatase. That these nucleotides might be specific signals for pho regulon expression was supported by the fact that the two nucleotides appearing under phosphate starvation induced the synthesis of alkaline phosphatase in repressed permeabilized wild-type cells under conditions of phosphate excess.     

Preparation and specificity of antibodies directed toward the ribose methylated nucleotide, 2'-O-methylguanosine 5'-monophosphate

Antibodies have been produced that recognize the ribosome methylated nucleotide, 2'-O-methylguanosine 5'-monophosphate, pGm. Specificity was tested in a radioimmunoassay. Other ribose methylated nucleotides, deoxyguanosine, and guanosine 5'-monophosphate exhibited negligible cross-reactivity. Elements of antibody recognition, in descending order of importance, were the parent base, the methylated ribose moiety, and the phosphate group.