guanosine-diphosphate and thymidine-5--triphosphate

Class II ribonucleotide reductases (RNR) catalyze the formation of an essential thiyl radical by homolytic cleavage of the Co-C bond in their adenosylcobalamin (AdoCbl) cofactor. Several mechanisms for the dramatic acceleration of Co-C bond cleavage in AdoCbl-dependent enzymes have been advanced, but no consensus yet exists. We present the structure of the class II RNR from Thermotoga maritima in three complexes: (i) with allosteric effector dTTP, substrate GDP, and AdoCbl; (ii) with dTTP and AdoCbl; (iii) with dTTP, GDP, and adenosine. Comparison of these structures gives the deepest structural insights so far into the mechanism of radical generation and transfer for AdoCbl-dependent RNR. AdoCbl binds to the active site pocket, shielding the substrate, transient 5'-deoxyadenosyl radical and nascent thiyl radical from solution. The e-propionamide side chain of AdoCbl forms hydrogen bonds directly to the α-phosphate group of the substrate. This interaction appears to cause a "locking-in" of the cofactor, and it is the first observation of a direct cofactor-substrate interaction in an AdoCbl-dependent enzyme. The structures support an ordered sequential reaction mechanism with release or relaxation of AdoCbl on each catalytic cycle. A conformational change of the AdoCbl adenosyl ribose is required to allow hydrogen transfer to the catalytic thiol group. Previously proposed mechanisms for radical transfer in B12-dependent enzymes cannot fully explain the transfer in class II RNR, suggesting that it may form a separate class that differs from the well-characterized eliminases and mutases.

Topics: Allosteric Regulation; Cobamides; Crystallography, X-Ray; Guanosine Diphosphate; Models, Molecular; Ribonucleotide Reductases; Thermotoga maritima; Thymine Nucleotides

E. coli ribonucleotide reductase (RNR), composed of the homodimeric subunits alpha2 and beta2, catalyzes the conversion of nucleotides to deoxynucleotides via complex radical chemistry. The radical initiation process involves a putative proton-coupled electron transfer (PCET) pathway over 35 A between alpha2 and beta2. Y356 in beta2 has been proposed to lie on this pathway. To test this model, intein technology has been used to make beta2 semi-synthetically in which Y356 is replaced with a DOPA-amino acid. Analysis of this mutant with alpha2 and various combinations of substrate and effector by SF UV-vis spectroscopy and EPR methods demonstrates formation of a DOPA radical concomitant with disappearance of the tyrosyl radical, which initiates the reaction. The results reveal that Y356 lies on the PCET pathway and demonstrate the first kinetically competent conformational changes prior to ET. They further show that substrate binding brings about rapid conformational changes which place the complex into its active form(s) and suggest that the RNR complex is asymmetric.

Topics: Adenosine Triphosphate; Amino Acid Substitution; Dihydroxyphenylalanine; Electron Spin Resonance Spectroscopy; Escherichia coli; Free Radicals; Guanosine Diphosphate; Kinetics; Models, Molecular; Oxidation-Reduction; Ribonucleotide Reductases; Spectrophotometry, Ultraviolet; Thymine Nucleotides; Tyrosine

Reduction of NDPs by murine ribonucleotide reductase (mRR) requires catalytic (mR1) and free radical-containing (mR2) subunits and is regulated by nucleoside triphosphate allosteric effectors. Here we present the results of several studies that refine the recently presented comprehensive model for the allosteric control of mRR enzymatic activity [Kashlan, O. B., et al. (2002) Biochemistry 41, 462-474], in which nucleotide binding to the specificity site (s-site) drives formation of an active R1(2)R2(2) dimer, ATP or dATP binding to the adenine site (a-site) drives formation of a tetramer, mR1(4a), which isomerizes to an inactive form, mR1(4b), and ATP binding to the hexamerization site (h-site) drives formation of an active R1(6)R2(6) hexamer. Analysis of the a-site D57N variant of mR1, which differs from wild-type mR1 (wt-mR1) in that its RR activity is activated by both ATP and dATP, demonstrates that dATP activation of the D57N variant RR arises from a blockage in the formation of mR1(4b) from mR1(4a), and provides strong evidence that mR1(4a) forms active complexes with mR2(2). We further demonstrate that (a) differences in the effects of ATP versus dATP binding to the a-site of wt-mR1 provide specific mechanisms by which the dATP/ATP ratio in mammalian cells could modulate in vivo RR enzymatic activity, (b) the comprehensive model is valid over a range of Mg(2+) concentrations that include in vivo concentrations, and (c) equilibrium constants derived for the comprehensive model can be used to simulate the distribution of R1 among dimer, tetramer, and hexamer forms in vivo. Such simulations indicate that mR1(6) predominates over mR1(2) in the cytoplasm of normal mammalian cells, where the great majority of RR activity is located, but that mR1(2) may be important for nuclear RR activity and for RR activity in cells in which the level of ATP is depleted.

Topics: Adenosine Triphosphate; Allosteric Regulation; Animals; Asparagine; Aspartic Acid; Cytidine Diphosphate; Deoxyadenine Nucleotides; Dimerization; Enzyme Activation; Guanosine Diphosphate; Kinetics; Light; Magnesium; Mice; Models, Chemical; Mutagenesis, Site-Directed; Protein Subunits; Recombinant Proteins; Ribonucleoside Diphosphate Reductase; Ribonucleotide Reductases; Scattering, Radiation; Substrate Specificity; Thymine Nucleotides; Tumor Cells, Cultured

Ribonucleotide reductase (RNR) is an essential enzyme in DNA synthesis, catalyzing all de novo synthesis of deoxyribonucleotides. The enzyme comprises two dimers, termed R1 and R2, and contains the redox active cysteine residues, Cys462 and Cys225. The reduction of ribonucleotides to deoxyribonucleotides involves the transfer of free radicals. The pathway for the radical has previously been suggested from crystallographic results, and is supported by site-directed mutagenesis studies. Most RNRs are allosterically regulated through two different nucleotide-binding sites: one site controls general activity and the other controls substrate specificity. Our aim has been to crystallographically demonstrate substrate binding and to locate the two effector-binding sites.. We report here the first crystal structure of RNR R1 in a reduced form. The structure shows that upon reduction of the redox active cysteines, the sulfur atom of Cys462 becomes deeply buried. The more accessible Cys225 moves to the former position of Cys462 making room for the substrate. In addition, the structures of R1 in complexes with effector, effector analog and effector plus substrate provide information about these binding sites. The substrate GDP binds in a cleft between two domains with its beta-phosphate bound to the N termini of two helices; the ribose forms hydrogen bonds to conserved residues. Binding of dTTP at the allosteric substrate specificity site stabilizes three loops close to the dimer interface and the active site, whereas the general allosteric binding site is positioned far from the active site.. Binding of substrate at the active site of the enzyme is structurally regulated in two ways: binding of the correct substrate is regulated by the binding of allosteric effectors and binding of the actual substrate occurs primarily when the active-site cysteines are reduced. One of the loops stabilized upon binding of dTTP participates in the formation of the substrate-binding site through direct interaction with the nucleotide base. The general allosteric effector site, located far from the active site, appears to regulate subunit interactions within the holoenzyme.

Topics: Allosteric Regulation; Amino Acid Sequence; Binding Sites; Conserved Sequence; Crystallography, X-Ray; Cysteine; Dimerization; Guanosine Diphosphate; Models, Chemical; Models, Molecular; Molecular Sequence Data; Oxidation-Reduction; Ribonucleotide Reductases; Sequence Alignment; Substrate Specificity; Thymine Nucleotides

Signal transduction via P2 purinergic receptors was investigated in HSG cells, a continuous cell line originally derived from an irradiated human salivary gland. Ligand specificity for nucleotide receptors in HSG cells was investigated with various nucleotides and their analogues. Inositol 1,4,5-trisphosphate (IP3) production was significantly increased by ATP, UTP and ATP gamma S. The ligand specificity of this effect agreed well with that of the P2U purinergic receptor. On the other hand, 45Ca2+ influx was stimulated by ATP, UTP > ATP gamma S, ADP, UDP > ADP beta S > AMPPNP, GTP, TTP > CTP, GDP, TDP, AMPPCP, AMPCPP. This ligand specificity of 45Ca2+ influx was much broader than IP3 production. Also pertussis and cholera toxin had no effect on both IP3 production and 45Ca2+ influx by ATP or UTP. 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) stimulates 45Ca2+ influx more effectively than IP3 formation. A 53-kDa membrane protein was photolabelled with [alpha-32P]Bz-ATP. This 53-kDa protein is a putative P2 purinergic receptor. In particular, the labelling was inhibited by a ligand profile that corresponded to that for 45Ca2+ influx. These findings suggest that nucleotides stimulate 45Ca2+ influx and IP3 formation by separate pathways via pertussis and cholera toxin-insensitive G proteins. Thus, in HSG cells, IP3 formation is coupled to the P2U subclass, while 45Ca2+ influx is coupled to another subclass, such as P2X, that regulates calcium channels.

Topics: Adenine Nucleotides; Adenosine Diphosphate; Adenosine Triphosphate; Adenylyl Imidodiphosphate; Affinity Labels; Calcium; Calcium Channels; Calcium Radioisotopes; Cell Line; Cholera Toxin; Cytidine Triphosphate; GTP-Binding Proteins; Guanosine Diphosphate; Humans; Inositol 1,4,5-Trisphosphate; Ligands; Membrane Proteins; Pertussis Toxin; Radiopharmaceuticals; Receptors, Purinergic; Salivary Ducts; Signal Transduction; Submandibular Gland; Substrate Specificity; Thionucleotides; Thymine Nucleotides; Uridine Triphosphate; Virulence Factors, Bordetella

Subunit B1 of Escherichia coli ribonucleotide reductase contains one type of allosteric binding site that controls the substrate specificity of the enzyme. This site binds the allosteric effector dTTP as well as other nucleoside triphosphates. Cross-linking of dTTP to protein B1 by direct photoaffinity labeling, as well as the isolation and sequence determination of the labeled tryptic peptide, has recently been reported [Eriksson, S., Sjöberg, B.-M., Jörnwall, H., & Carlquist, M. (1986) J. Biol. Chem. 261, 1878-1882]. In this study, we have further purified the dTTP-labeled peptide and characterized it using UV spectroscopy. Two types of dTTP-cross-linked peptide were found: one having an absorbance maximum at 261 nm typical for a dTTP spectrum, i.e., containing an intact 5,6 double bond, and one minor form with low absorbance at 261 nm. In both cases, the same amino acid composition was found, corresponding to the peptide Ser291-X-Ser-Gln-Gly-Gly-Val-Arg299 in the B1 sequence with X being Cys-292 cross-linked to dTTP. Isotope labeling experiments revealed that one proton in the 5-methyl group of thymine was lost during photoincorporation. Therefore, the cross-linking occurs via the 5-methyl group to Cys-292 in a majority of incorporated dTTPs, but a second, possibly 5,6-saturated form of incorporated nucleotide was also detected. The reasons for the high stereospecificity of the reaction and the possible structure of the allosteric site of protein B1 are discussed.

Topics: Affinity Labels; Allosteric Site; Escherichia coli; Guanosine Diphosphate; Macromolecular Substances; Phosphorus Radioisotopes; Ribonucleotide Reductases; Thymine Nucleotides; Tritium; Ultraviolet Rays

The 5'-mono-, di- and triphosphate derivatives (N3dTMP, N3dTDP and N3dTTP respectively) of 3'-azidothymidine (N3dThd), a new drug for the treatment of the acquired immune deficiency syndrome (AIDS), were synthesized. The abilities of these analog nucleotides to mimic the effector properties of the corresponding thymidine nucleotides with human ribonucleotide reductase were studied. Surprisingly, the mode of inhibition of CDP reduction by dTTP and dTDP was found to be competitive versus CDP. The Ki values were 22 and 78 microM respectively. Inhibition by N3dTTP and N3dTDP was considerably weaker, with Ki values of 1200 and 550 microM. Neither dTMP nor N3dTMP produced significant inhibition at concentrations up to 500 microM. dTTP was an essential activator for GDP reduction. In the presence of the accessory activator, ATP, the activation constant for dTTP was 7.8 microM. N3dTTP was neither an activator of GDP reduction nor an inhibitor of the activation by dTTP. In view of the intracellular concentrations of these analog nucleotides reached after incubations with N3dThd [Furman et al., Proc. natn. Acad. Sci. U.S.A. 83, 8333 (1986)] and the weakness of their interactions with ribonucleotide reductase, it is unlikely that the antiviral or toxic effects of N3dThd can be attributed to direct effects on this enzyme. The possible indirect effects caused by alterations in the pools of the natural effectors are discussed.

Topics: Antiviral Agents; Cytidine Diphosphate; Guanosine Diphosphate; Humans; Kinetics; Nucleotides; Oxidation-Reduction; Ribonucleotide Reductases; Thymidine; Thymine Nucleotides; Zidovudine

Topics: Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Escherichia coli; Guanosine Diphosphate; Macromolecular Substances; Photochemistry; Ribonucleotide Reductases; Thymine Nucleotides