Nucleotide Complexes Research Articles

Molecular complexes of nucleosides and nucleotides with a monomeric cationic porphyrin and some of its metal derivatives.

Tetrakis(4-7V-methylpyridyl)porphine (H2TMpyP) and a number of its metal derivatives interact extensively with mononucleotides and mononucleosides in aqueous solution. The complexes formed are of a stacking-type involving extensive overlap of the -systems of the porphyrin and purine or pyrimidine bases. Coulombic attractions help stabilize the complexes but there is no evidence for ligation of the bases to axial sites of the metalloporphyrins. Stability constants determined via NMR and spectrophotometric titrations are larger for purine bases than pyrimidines with a given porphyrin derivative. More dramatic influences on stability result from changing porphyrins. Porphyrins having no axial ligands (e.g., metal-free copper(II), palladium(II), and nickel(II) derivatives) or one axial ligand (Zn(II)) produce much larger interactions with a given nucleotide or nucleoside than do metalloporphyrins having two axial ligands (e.g., Mn(III), Fe(III), or Co(III)). The kinetics of the interaction of H2TMpyP with 2'-deoxyadenosine S'-monophosphate (dAMP) were studied via the laser raman temperature-jump method. The measured rate constants are consistent with a simple stacking model for the interaction.

Kinetic mechanism of 1-N6-etheno-2-aza-ATP hydrolysis by bovine ventricular myosin subfragment 1 and actomyosin subfragment 1. The nucleotide binding steps.

The large change in fluorescence emission of 1-N6-etheno-2-aza-ATP (epsilon-aza-ATP) has been used to investigate the kinetic mechanism of etheno-aza nucleotide binding to bovine cardiac myosin subfragment 1 (myosin-S1) and actomyosin subfragment 1 (actomyosin-S1). The time course of nucleotide fluorescence enhancement observed during epsilon-aza-ATP hydrolysis is qualitatively similar to the time course of tryptophan fluorescence enhancement observed during ATP hydrolysis. In single turnover experiments, the nucleotide fluorescence rapidly increases to a maximum level, then decreases with a rate constant of 0.045 s-1 to a final level, which is about 30% of the maximal enhancement; a similar fluorescence enhancement is obtained by adding epsilon-aza-ADP to cardiac myosin-S1 or actomyosin-S1 under the same conditions (100 mM KCl, 10 mM 4-morpholinepropanesulfonic acid, 5 mM MgCl2, 0.1 mM dithiothreitol, pH 7.0, 15 degrees C). The kinetic data are consistent with a mechanism in which there are two sequential (acto)myosin-S1 nucleotide complexes with enhanced nucleotide fluorescence following epsilon-aza-ATP binding. The apparent second order rate constants of epsilon-aza-ATP binding to cardiac myosin subfragment 1 and actomyosin subfragment 1 are 2-12 times slower than those for ATP. Actin increases the rate of epsilon-aza-ADP dissociation from bovine cardiac myosin-S1 from 1.9 to 110 s-1 at 15 degrees C which can be compared to 0.3 and 65 s-1 for ADP dissociation under similar conditions. Although there are quantitative differences between the rate and equilibrium constants of epsilon-aza- and adenosine nucleotides to cardiac actomyosin-S1 and myosin-S1, the basic features of the nucleotide binding steps of the mechanism are unchanged.

Investigation of the preferred Mg(II)‐adenine‐nucleotide complex at the active site of ectonucleotidases in intact vascular cells using phosphorothioate analogues of ADP and ATP

In the presence of Mg2+ the ecto-(nucleoside diphosphatase) on intact vascular endothelial or smooth muscle cells in culture selectively catabolizes the PS diastereoisomer of adenosine 5'-[alpha-thio]diphosphate, (PS)-ADP [alpha S], and the ecto-(nucleoside triphosphatase) selectively catabolizes the PS isomer of adenosine 5'-[beta-thio]triphosphate, (PR)-ATP[beta S], but exhibits no selectivity towards ATP[alpha S] isomers. In the presence of Cd2+ selectivity to ADP[alpha S] and to ATP[beta S] isomers is reversed; in the presence of Co2+, selectivity is lost. We conclude that each enzyme preferentially recognises the lambda (screw-sense) bidentate Mg(II)-nucleotide complex at its active site.

The magnesium protein interaction in nucleotide complexes of phos-phoglycerate kinase

The X-ray structure determination of yeast phosphoglycerate kinase and subsequent substrate binding studies have helped to define the binding sites for the triose and nucleoside phosphate substrates. This communication deals with one feature of the binding site—the location of an aspartic acid residue close to the phosphoryl binding site of the nucleotide substrate—and relates this and other structural features of the active site to the properties of this enzyme as deduced from nuclear magnetic resonance studies.

Molecular movements promoted by metal nucleotides in the heavy-chain regions of myosin heads from skeletal muscle

Molecular movements generated in the heavy-chain regions (27-50-20(× 10 3) M r) of myosin S1 ‡ ‡ Abbreviations used: S1, chymotryptic subfragment-1 of myosin; AMPPNP, 5′-adenilyl imidodiphosphate; PP i, pyrophosphate; HMM, heavy meromyosin; SDS, sodium dodecyl sulfate; LC, light chain; IAEDANS, N-iodoacetyl- N′-(5-sulfo-1-napthyl)ethylenediamine. on interaction with nucleotides ATP, AMPPNP, ADP and PP i were investigated by limited proteolysis of several enzyme-metal nucleotide complexes in the absence and presence of reversibly bound and crosslinked F-actin. The rate and extent of the nucleotidepromoted conversion of the NH 2-terminal 27 × 10 3 M r and 50 × 10 3 M r segments into products of 22 × 10 3 M r and 45 × 10 3 M r, respectively, were estimated to determine the amplitude of the molecular movements. The 22 × 10 3 M r peptide was identified by amino acid sequence studies as being derived from cleavage of the peptide bond between Arg and Ile (at position 23 to 24). The 45 × 10 3 M r, peptide, previously shown to represent the NH 2-terminal part of the 50 × 10 3 M r region, would be connected to the adjacent C-terminal 20 × 10 3 M r region by a pre-existing loop segment of about 5 × 10 3 M r; the proteolytic sensitivity of the latter region is increased particularly by nucleotide binding. The tryptic reaction proved to be a sensitive indicator of the conformational state of the liganded heavy chain as the rate of peptide bond cleavage in the two regions is dependent on the nature of the bound ligand; it decreases in the order: ATP > AMPPNP > ADP > PP i. It depends also on the nature of the metal present, Mg 2+ and Ca 2+ being much more effective than K +. Binding of F-actin to the S1-MgAMPPNP complex affords significant protection against breakdown of 27 × 10 3 M r and 50 × 10 3 M r peptides, but with concomitant hydrolysis of the 50 × 10 3 M r −20 × 10 3 M r junction. Additionally, interaction of MgATP with HMM modulates the tryptic fission of the S1-S2 region. The overall data provide a molecular support for the two-state model of the myosin head and emphasize the involvement of the 50 × 10 3 M r unit in the mechanism of coupling between the actin and nucleotide binding sites.

Metal-nucleotide structural characteristics during catalysis by beef heart mitochondrial F1.

This study examined the nature of the metal-nucleotide complexes which serve as substrates, products, and intermediates in the beef heart mitochondrial ATPase reaction. The two methods employed involved the use of phosphorothioate ATP analogs as substrates in the presence of Mg2+ or Cd2+ and the use of substitution inert Cr X ATP complexes (the isolated diastereomers of the bidentate complexes) along with the newly synthesized Cr X ITP complexes as inhibitors of both the F1-ATPase and F1-ITPase activities. Little stereoselectivity was observed in the inhibition of F1-ATPase and F1-ITPase activities by the isolated diastereomers of beta,gamma-bidentate CrATP, while the inhibition by the delta,alpha,beta-bidentate CrADP diastereomer was greater than that of the lambda epimer. gamma-Monodentate CrITP was a weak inhibitor of both the ATPase and ITPase activities, whereas beta,gamma-bidentate CrITP failed to show any inhibition at all up to a concentration of 3.2 mM. When adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) was used as the substrate, (VmSp]/(Vm(Rp] with Mg2+ present was 2.7 at 31 degrees C and 3.5 at 13 degrees C. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Mg2+ present were 15.3 at 31 degrees C and 73.3 at 13 degrees C. With Cd2+ present, the (Vm(Sp]/(Vm(Rp] ratios were 0.81 and 0.65 at 31 and 13 degrees C, respectively. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Cd2+ present were 1.17 at 31 degrees C and 1.34 at 13 degrees C. The large activation energy observed for the isomers of CdATP beta S was not observed for MgATP beta S, MgATP, or CdATP. The Vm for Cd adenosine 5'-O-thiotriphosphate (ATP gamma S) hydrolysis was the largest of all the metal-phosphorothioate nucleotide complexes, while that for MgATP gamma S was the smallest. The results are interpreted in terms of a catalytic model for F1-catalyzed nucleotide hydrolysis describing metal-nucleotide chelation during the reaction.

Nature of the terbium(III) excitation band observed near 300 .mu.m in certain nucleotide or nucleic acid complexes

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNature of the terbium(III) excitation band observed near 300 .mu.m in certain nucleotide or nucleic acid complexesAllen Rudman, Sergio Paoletti, and Harry G. BrittainCite this: Inorg. Chem. 1985, 24, 9, 1283–1285Publication Date (Print):April 1, 1985Publication History Published online1 May 2002Published inissue 1 April 1985https://doi.org/10.1021/ic00203a003RIGHTS & PERMISSIONSArticle Views83Altmetric-Citations9LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (417 KB) Get e-Alerts Get e-Alerts

A spectrophotometric resin competition procedure to determine dissociation constants for metal ion-nucleotide complexes

The resin competition method for determining the dissociation constants of metal ion-nucleotide complexes was modified to take into account the fact that some metal nucleotide complexes are anionic and thus, like the free nucleotide, will bind to the resin. A simple, rapid spectrophotometric titration procedure is given for the determination of both the metal ion-nucleotide complex dissociation constant and the ratio of the affinities of the nucleotide and its complex for the resin.

Calcium regulation of molluscan myosin ATPase in the absence of actin.

In the myosin-linked regulatory mechanism typified by the molluscan scallop adductor muscle, contraction is controlled by Ca2+ binding to sites on the thick filament protein, myosin. The regulatory light chains of myosin heads are involved directly in this mechanism and early studies suggested that, in the absence of Ca2+, these subunits prevent the interaction of a myosin-adenosine nucleotide complex with the actin-containing thin filament. Subsequently, Ashiba et al. reported that the steady-state ATPase of molluscan myosin exhibits a limited degree of Ca2+ activation in the absence of actin. Recently, however, we have shown that steady-state ATPase activity in relaxing conditions is dominated by the unregulated molecules in the myosin preparation. Single-turnover kinetic methods are required to monitor the highly suppressed ATPase activity of the regulated population. Using the latter approach, we report here that scallop myosin ATPase is reduced about 100-fold on removal of Ca2+. The regulatory light chains maintain the relaxed state via conformational changes which suppress the product release steps, irrespective of the presence of actin.

Specificity of the functional interactions of the beta-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles.

We have assessed the functional interactions of two pure receptor proteins with three different pure guanine nucleotide regulatory proteins in phosphatidylcholine vesicles. The receptor proteins are the guinea pig lung beta-adrenergic receptor (beta AR) and the retinal photon receptor rhodopsin. The guanine nucleotide regulatory proteins were the stimulatory (Ns) and inhibitory (Ni) proteins of the adenylate cyclase system and transducin (T), the regulatory protein from the light-activated cyclic GMP phosphodiesterase system in retinal rod outer segments. The insertion of Ns with beta AR in lipid vesicles increases the extent of binding of [35S] GTP gamma S to Ns and in parallel, the total GTPase activity. However, there is little change in the actual rate of catalytic turnover of GTPase activity (defined as mol of Pi released/min/mol of Ns-guanine nucleotide complexes). Enhancement of this turnover rate requires the beta-agonist isoproterenol and is accounted for by an isoproterenol-promoted increase in the rate and extent of [35S]GTP gamma S binding to Ns. The co-insertion of the beta AR with Ni or transducin results in markedly lower stimulation by isoproterenol of both the GTPase activity and [35S]GTP gamma S binding to these nucleotide regulatory proteins indicating that their preferred order of interaction with beta AR is Ns much greater than Ni greater than T. This contrasts with the preferred order of interaction of these different nucleotide regulatory proteins with light-activated rhodopsin which we find to be T approximately equal to Ni much greater than Ns. Nonetheless the fold stimulation of GTPase activity and [35S]GTP gamma S binding in T, induced by light-activated rhodopsin, is significantly greater than the "fold" stimulation of these activities in Ni. This reflects the greater intrinsic ability of Ni to hydrolyze GTP and bind guanine nucleotides (at 10 mM MgCl2, 100-200 nM GTP or [35S] GTP gamma S) compared to T. The maximum turnover numbers for the rhodopsin-stimulated GTPase in both Ni and T are similar to those obtained for isoproterenol-stimulated activity in Ns. This suggests that the different nucleotide regulatory proteins are capable of a common upper limit of catalytic efficiency which can best be attained when coupled to the appropriate receptor.

Absence of direct metal–nucleotide interaction in the crystal structure of the ternary system, [Cu(bim)(H2O)5]2+[5′-IMP]2–·3H2O (bim = benzimidazole; 5′-IMP = inosine 5′-monophosphate)

The unusual absence of direct metal-nucleotide binding in a metal-6-oxopurine nucleotide complex is demonstrated for the first time in the structure of $[Cu(bim)(H_2O)5]^{2+}[5'-IMP-.3]^{2-}3H_2O$ (bim = benzimidazole; 5'-IMP = inosine 5'-monophosphate); the nucleotide base stacks with the bim ligand and the phosphate oxygens are involved in hydrogen bonds with the co-ordinated water molecules.

The synthesis and characterization of nucleotide complexes involving an asymmetric metal

The synthesis of cobalt and chromium complexes of H4ATP and H4GTP in which the metal is asymmetric are reported. These compounds were characterized by visible spectroscopy, fast atom bombardment mass spectroscopy (FAB MS), and 31P NMR. The mass spectral data allow identification of the complexes to be made from ions in the molecular weight region. The effect of an asymmetric metal greatly alters the appearance of the 31P NMR spectra in comparison to complexes which do not have this feature. Complexes of uridine diphosphoglucose, UDPG, are also reported. The effect of an asymmetric metal ion on the chromatographic and spectral properties of the complexes are discussed.

Guanosine nucleotide binding by highly purified Ha-ras-encoded p21 protein produced in Escherichia coli.

High-level expression of the p21 protein product of the BALB murine sarcoma virus v-ras gene (similar to the product of the Harvey murine sarcoma virus v-Ha-ras gene) has been reported recently, and highly purified preparations of this protein have been obtained. We used a nitrocellulose filter assay for measuring the binding of GDP and GTP to the purified protein. Previously p21 antibodies had been used to precipitate p21-guanosine nucleotide complexes from crude extracts containing the protein. Using the filter assay, we find that the v-Ha-ras gene product binds [3H]GDP stoichiometrically. The binding is time-dependent and is faster at 30 degrees C than at 0 degrees C. Optimum binding is obtained in the presence of dithiothreitol and magnesium ions and at pH 7.4. In terms of its GDP binding activity, p21 is heat stable and pronase sensitive. The dissociation constants (Kd) of p21 for [3H]GDP and [3H]GTP, determined by Scatchard analysis, are 6 X 10(-8) M and 2.5 X 10(-8) M, respectively.

Stability constants of Mg2+ and Cd2+ complexes of adenine nucleotides and thionucleotides and rate constants for formation and dissociation of MgATP and MgADP.

Stability constants for the Mg2+ and Cd2+ complexes of ATP, ADP, ATP alpha S, ATP beta S, and ADP alpha S have been determined at 30 degrees C and mu = 0.1 M by 31P NMR. Besides being of the utmost importance for determining species distributions for enzymatic studies, these constants allow an estimation of the preference of Cd2+ for sulfur vs. oxygen coordination in phosphorothioate complexes. Stability constants for Mg2+ complexes decreases when sulfur replaces oxygen (log K: ADP, 4.11; ADP alpha S, 3.66; ATP, 4.70; ATP alpha S, 4.47; ATP beta S, 4.04) because of (a) a statistical factor resulting from the loss of one potential phosphate oxygen ligand and (b) either an alteration in the charge distribution between oxygen and sulfur or destabilization of the chelate ring structure by loss of an internal hydrogen bond between an oxygen of coordinated phosphate and metal-bound water. Cd2+ complexes with sulfur-substituted nucleotides are more stable than those without sulfur (log K: ADP, 3.58; ADP alpha S, 4.95; ATP, 4.36; ATP alpha S, 4.42; ATP beta S, 5.44) because of the preferential binding of Cd2+ to sulfur rather than oxygen, which we estimate to be approximately 60 in CdADP alpha S and CdATP beta S. The proportion of tridentate coordination is estimated to be 50-60% in MgATP and MgATP beta S, approximately 27% in MgATP alpha S, approximately 16% in CdATP or CdATP beta S, but approximately 75% in CdATP alpha S. By analysis of the data of Jaffe and Cohn [Jaffe, E. K., & Cohn, M. (1979) J. Biol. Chem. 254, 10839], we conclude that the preference for oxygen over sulfur coordination to ATP beta S is 31 000 for Mg2+, 3100-3900 for Ca2+, and 158-193 for Mn2+. Proton NMR demonstrates that bidentate Cd2+ complexes form intramolecular chelates with the N-7 of adenine while Mg2+ nucleotides and the tridenate CdATP alpha S do not. An analysis of the 31P NMR line widths shows that the rate constants for dissociation of MgADP and MgATP are both 7000 s-1 while the association rate constants are 7 X 10(7) and 4 X 10(8) M-1 s-1, respectively. The observed dependence of the line width on nucleotide concentration is best explained by a base-stacking model at nucleotide concentrations above 5 mM.

The crystal and molecular structure of [Co(NH 3) 5PO 4]·3H 2O, a Na +-K + ATPase probe

The title compound, Co(NH 3) 5PO 4, prepared by a modified literature procedure, was used to study the inhibition of Na +-K + ATPase and to serve as a structural model for ML 4(nucleotide) complexes. The structure was determined by single crystal X-ray diffraction techniques. The crystals are monoclinic, space group P2 1/n, a = 8.638(3), b = 14.517(2), c = 9.145(2) Å, and β = 112.71(2)°. The structure, solved by the heavy atom method to an R value of 3.3% for 1924 reflections, consists of a slightly distorted octahedron with the cobalt bound to the five amines and a monodentate phosphate. Solution structural data is taken from 31P NMR measurements. From comparison with other metal phosphate complexes it is concluded that multiple monodentate coordination of a di- or triphosphate closely resembles the coordination of a monophosphate This is based on the similarity of the MO bond angle which is 129.6° in the present example.

Fast atom bombardment mass spectrometry of metal nucleotide complexes.

The fast atom bombardment mass spectrometry of the beta-gamma complexes of ATP and GTP with cobalt and chromium are reported. Spectra were recorded in the positive ion mode. Ions in the molecular weight region allow identification of the complexes to be made.

The characterization of vanadate-trapped nucleotide complexes with spin-labelled myosins.

The properties of spin-labelled myosin, prepared from rabbit skeletal and scallop adductor muscle, on forming a long-lived complex with ADP and vanadate (M.ADP.Vi), have been investigated. In the case of an iodoacetamide-based label attached to rabbit myosin or subfragment 1, M.ADP.Vi formation is characterized by a marked increase in the mobility of the probe, similar to that seen during steady-state ATPase activity. Hence, this complex appears to be a good analogue of the M**ADP.Pi state. The kinetics of M.ADP.Vi formation were determined by following the electron paramagnetic resonance (e.p.r.) signal with time and were analysed according to the scheme: (formula; see text) After correction for Vi polymerization, K'4 = 3.2 X 10(-4)M, k'-3 = 8.7 X 10(-3) s-1 and k'3 = 1.5 X 10(-4) s-1. The major effect of spin-labelling the reactive SH1 thiol is to increase k'3, so that M.ADP.Vi dissociates over a period of hours rather than days. In contrast, a maleimide-based spin-label attached to rabbit myosin does not exhibit a large change in mobility, on formation of the M.ADP.Vi complex. However, the small change observed in both the conventional and saturation transfer spectra questions the assumption that this probe is completely insensitive to librational motion during ATPase activity. The immobilized spectrum of the iodoacetamide-based spin label attached to scallop myosin is insensitive to M.ADP.Vi formation in the presence or absence of Ca2+. Under these conditions, the label appears to reflect gross head motion and hence this observation lends no support to the idea that, in the myosin-linked regulatory system, Ca2+ operates by controlling the flexibility of the subfragment 1-subfragment 2 joint.

Covalent Interaction of Proteins and Nucleic Acids. Synthetic and Natural Nucleotide-Peptides

Abstract The review reports data on the occurrence, structure and functions of covalent nucleotide and NA-protein complexes. Methods of synthesis of model nucleotide-peptides with phosphoester and phosphoamide bonds and their hydrolytic properties are discussed. Separate sections are devoted to methods of specific cleavage of phosphoamide and phosphoester bonds in nucleotide-peptides and their application in biochemistry.

Chemical Crosslinking of Myosin Subfragment-1 to F-Actin in the Presence of Nucleotide1

F-Actin and myosin subfragment-1 (S-1) were covalently crosslinked in the absence and presence of nucleotides, adenyl-5'-yl imidodiphosphate (AMPPNP), ATP, and ADP, at various KCl concentrations (0-0.5 M), using a zero-length crosslinker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). The rate of production of the covalent acto-S-1 complex was almost proportional to the amount of the acto-S-1(-nucleotide) complex existing in the reaction medium. However, the Mg-ATPase activity of the covalent acto-S-1 complex thus produced was affected by the presence of nucleotides. When S-1 was crosslinked to F-actin in the absence of nucleotide at 0-0.5 M KCl, the Mg-ATPase activity of S-1 was enhanced from 0.1-0.3 to 12-15 s-1. The Mg-ATPase activity of covalent complex produced in the presence of AMPPNP at 0 M KCl was 13 s-1 which was equal to that produced in the absence of nucleotide. The activity decreased with increasing KCl concentration of the crosslinking medium, reaching 6 s-1 at 0.5 M KCl. Covalent acto-S-1 complex was also produced when acto-S-1-ADP-P was formed during ATP hydrolysis. The Mg-ATPase activity of the covalent acto-S-1 complex crosslinked during ATP hydrolysis at 0 M KCl was about 5 s-1 which was about half of that obtained in the absence of nucleotide, and it decreased to about 2 s-1 at 0.15 M KCl of crosslinking medium. In contrast, the acto-S-1-ADP complex gave a covalent complex which was indistinguishable in its extent and Mg-ATPase activity from that obtained in the absence of nucleotide. These results suggest that the structures of acto-S-1-AMPPNP and acto-S-1-ADP-P complexes are different from those of acto-S-1 and acto-S-1-ADP complexes.

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.