Nucleotide Complexes Research Articles

Synergism between different metal ions in the dephosphorylation of adenosine 5′-triphosphate (ATP) in mixed metal ion/ATP systems, and influence of a decreasing solvent polarity (dioxane-water mixtures) on the dephosphorylation rate. Effects of Mg2+, Na+, and NH4+ ions

The initial rates of the dephosphorylation (i.e., v 0 = d[PO 4 ]/dt) of adenosine 5′-triphosphate (ATP) (=10 −3 M) in the mixed metal ion systems Cu 2+ /ATP/Mg 2+ , Ni 2+ , Zn 2+ , or Cd 2+ with the ratios 1:1:1 and 1:1:5 have been measured and compared at pH 5.5 and 50°C (I = 0.1, NaClO 4 ) with the corresponding binary ATP/M 2+ 1:2 and 1:6 systems. The remarkable result is that addition of Mg 2+ to a Cu 2+ /ATP 1:1 system accelerates the dephosphorylation rate significantly more than the same amounts of Mg 2+ accelerate the reaction in a Mg 2+ /ATP 1:1 system. The same synergism, based also on the Cu 2+ /ATP 1:1 system, is observed with Ni 2+ , but not with Zn 2+ and Cd 2+ . This observation is attributed to the formation of a prereactive state in the Cu 2+ /ATP 1:1 system, i.e., of a [Cu(ATP)] 2 4− dimer which involves purine-stacking and a Cu 2+ /N-7 interaction; the inherent reactivity in this dimer may be triggered by the addition of Mg 2+ or Ni 2+ . In Zn 2+ or Cd 2+ /ATP 1:1 systems also a prereactive state is formed and therefore no synergism is observed in a comparison with the corresponding mixed Cu 2+ systems. In agreement herewith, there is a rather pronounced synergism in the Zn 2+ /ATP Mg 2+ system at pH 7.5, and a somewhat smaller one under the same conditions in the Zn 2+ /ATP/Na + system. In the latter system the synergism may be considerably favored by reducing the solvent polarity, i.e., by changing the solvent from water to 50% (v/v) dioxane-water; similar effects, though less pronounced, are observed with Zn 2+ /UTP. In connection with the solvent effects it is recalled that the polarity in the active-site cavities of enzymes is also lower than in the bulk water. By experiments with ATP and Mg 2+ /ATP systems it is shown that Na + and NH 4 + have corresponding effects; this observation is important regarding cationic side chains of amino acid residues in proteins. Some further implications of the present results for biosystems are also indicated. The phosphate groups in TNP are labeled as α, β, and γ, where the latter refers to the terminal phosphate group (Fig. 1). If nothing else is specified, the formula PO 4 reperesents all related species which may be present in solution, i.e., H 3 PO 4 , H 2 PO 4 − , HPO 4 2− , and PO 4 3− . The term “dephosphorylation” is used for the transfer of a phosphoryl group to a water molecule; the term “hydrolysis” can also refer to this, but for the most part we use it in connection with the formation of hydroxo complexes of metal ions. The terms monomeric or dimeric complexes mean that one or two NTP 4− together with at least the corresponding equivalents of M 2+ are within the considered complex; hence, e.g., M 2 (NTP) is a monomeric (but dinuclear) nucleotide complex whereas [M(NTP)] 2 4− is a dimeric one.

Stereochemically controlled ligands influence atropisomerization of platinum(II) nucleotide complexes. Evidence for head-to-head and stable .LAMBDA. head-to-tail atropisomers

Stereochemically controlled ligands influence atropisomerization of platinum(II) nucleotide complexes. Evidence for head-to-head and stable .LAMBDA. head-to-tail atropisomers

31P NMR studies of enzyme-bound substrate complexes of yeast 3-phosphoglycerate kinase: III. Two ADP binding sites and their Mg(II) affinity; effects of vanadate and arsenate on enzymic complexes with ADP and 3-P-glycerate

31P nuclear magnetic resonance (NMR) measurements (at 121.5 MHz and 5°C) were made on complexes of 3-phosphoglycerate kinase with ADP and 3-P-glycerate. Addition of Mg(II) to E-ADP shifts the α-Psignal downfield by 3.8 ppm such that the α-P signal superimposes that for β-P(E·MgADP). Such a shift is atypical among the Mg(II)-nucleotide complexes with other ATP-utilizing enzymes. This shift allowed the determination that enzyme bound ADP is saturated with Mg(II) for [Mg(II)]/[ADP] = 3.0-similar to that reported for ATP complexes with this enzyme (B. D. Ray and B. D. Nageswara Rao, Biochemistry 27, 5574 (1988)). This parallel behavior suggests that ADP binds at two sites on the enzyme as does ATP with disparate Mg(II) affinities. 31P relaxation times in E·MnADP·vanadate-3-P-glycerate and E·CoADP·vanadate·3-P-glycerate complexes indicate that these are long-lived, tightly bound complexes. 31P chemical shift measurements on diamagnetic complexes (with Mg(II)) revealed three signals in the 2–5 ppm region (attributable to 3-P-glycerate) only upon addition of all the components necessary to form the E·MgADP·vanadate·3-P-glycerate complex. Subsequent sequestration of Mg(II) from the complex with excess EDTA reversed the Mg(II) induced effects on the ADP signals but did not cause coalescence of the three signals seen in the 2–5 ppm region. Addition of excess sulfate to dissociate these complexes from the enzyme resulted in a single resonance of 3-P-glycerate. The use of arsenate in place of vanadate yielded very similar results. These results suggest that, in the presence of MgADP, vanadate or arsenate, and 3-P-glycerate, the enzyme catalyzed the formation of multiple structurally distinguishable complexes that are stable on the enzyme and labile off the enzyme.

Reactions of iron(II) nucleotide complexes with hydrogen peroxide

The rate constants for the reactions of hydrogen peroxide with ferrous complexes of ATP, ADP, UTP, citrate and pyrophosphate were measured at pH 7.2. These ligands are potential chelators of iron(II) in the low-molecular weight iron pool that may catalyze oxidative degradation of tissues. The second-order rate constants range from 5.5 × 10 3 M −1s −1 (UTP) to 1 × 10 5 M −1s −1 (pyrophosphate) at pH 7.2. The kinetic dependences of the ATP reaction are consistent with a mechanism involving a ‘ferryl’ (Fe IV) transient which decays to the hydroxyl radical and ferric ATP.

Reversed-phase high-performance liquid chromatographic study of the formation of complexes of nucleotides and oligonucleotides with Lu(III)

Reversed-phase high-performance liquid chromatographic study of the formation of complexes of nucleotides and oligonucleotides with Lu(III)

Effect of ethylene glycol on the interaction of different myosin subfragment 1.cntdot.nucleotide complexes with actin

To elucidate the structure of the cross-bridge intermediates in the actomyosin ATPase cycle, several laboratories have added both ethylene glycol and AMP-PNP to muscle fibers. These studies suggested that ethylene glycol shifts the structure of myosin.AMP-PNP toward the weak-binding conformation, i.e., toward the structure of myosin.ATP. Since only the weak-binding conformation of myosin subfragment 1 (S-1) binds with no apparent cooperativity to the troponin-tropomyosin-actin complex (regulated actin), we used this as a probe to examine the conformation of various S-1.nucleotide complexes in ethylene glycol. Our results show that ethylene glycol markedly weakens the binding strength of S-1, S-1.ADP, and S-1.AMP-PNP to actin but has almost no effect on the binding strength of S-1.ATP. As in muscle fibers, at 40% ethylene glycol, the binding strength of S-1.AMP-PNP to actin becomes very similar to the binding strength of S-1.ATP. In the presence of troponin-tropomyosin, the binding of S-1.AMP-PNP to actin shows no apparent cooperativity in 40% ethylene glycol. Therefore, our results confirm that ethylene glycol shifts the structure of the myosin.AMP-PNP toward the weak-binding conformation. However, our results also suggest that ethylene glycol has a direct effect on the regulated actin complex. This is shown by the fact that ethylene glycol markedly increases the cooperative binding of S-1.ADP to regulated actin both in the presence and in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Self-association of nucleotides

The self-association of nucleosides decreases within the series adenosine greater than guanosine greater than inosine greater than cytidine approximately uridine. The same trend is observed for the corresponding nucleotides, though less pronounced, as the charge effect governs series like adenosine much greater than AMP2- greater than ADP3- approximately greater than ATP4-. Protonation of adenosine considerably reduces its self-stacking tendency: this is different with ATP4-, where a maximum is reached for H2(ATP)2- caused by additional ionic interactions in the [H2(ATP)]2(4-) dimer. Metal ion coordination may promote self-association, e.g., of ATP4- via (mainly) charge neutralization (Mg2+) and the formation of intermolecular bridges in dimeric stacks (Zn2+, Cd2+). These results allow definition of conditions with negligible self-association and thus the determination of the stability and structure of monomeric nucleotide complexes in aqueous solution, e.g., quantification of macrochelate formation in M(ATP)2- complexes. Some biological implications of the results are indicated.

Synthetic site-directed ligands

Complexes of nucleotides, peptides and aromatic hapten-like compounds with immunoglobulin fragments were studied by X-ray analysis. After tri- or hexanucleotides of deoxythymidylate were diffused into triclinic crystals of a Fab (BV04-01) with specificity for single-stranded DNA, extensive changes were detected throughout the structure of the protein. The Fab co-crystallized with a tri- or pentanucleotide in a different space group (monoclinic), an observation sometimes correlated with alterations in the structure of the 'native' protein. Structural analyses of the co-crystals are in progress for direct comparisons with the unliganded Fab. In crystals of a human (Mcg) Bence-Jones dimer, synthetic opioid peptides, chemotactic peptides or dinitrophenyl (DNP) derivatives could be diffused into a large conical binding cavity. The conformations of both the ligand and the protein were usually altered during the binding process. At the base of the cavity tyrosine residues could be displaced like trap-doors to permit entry of some opioid peptides and DNP compounds into a deep binding pocket. In co-crystals of the dimer and bis(DNP)lysine, two ligand molecules were bound in tandem, one in the main cavity and the second in the deep pocket. One ligand adopted an extended conformation, with the epsilon-DNP ring near the floor of the main cavity and the alpha-DNP group in solvent outside the binding site. There were no significant conformational changes in the protein. In contrast, the second ligand was very compact, with DNP rings immersed in the deep pocket, and the binding site was expanded to accommodate the oversized ligand. Peptides designed to be specific for the main cavity were incrementally constructed from minimal binding units by M. Geysen, G. Trippick, S. Rodda and their colleagues. A pentapeptide optimized for binding by this method was diffused into a crystal of the dimer and found by Fourier difference analysis to lodge exclusively in the main cavity as predicted. Binding regions in the BV04-01 Fab and the Mcg dimer were markedly different in size and shape. The Fab had a groove-type site, in which a layer of sidechains acted like a false floor over regions analogous to the cavity and deep pocket of the Bence-Jones dimer.

Uranyl Ion as a Highly Effective Catalyst for Internucleotide Bond Formation

Abstract Uranyl ion catalyzes the polymerization of adenosine-5′-phosphorimidazolide very efficiently in aqueous solution. Up to 95% of the input adenosine-5′-phosphorimidazolide is converted to oligoadenylates from dimer to hexadecamer when 1 mM of UO22+ is used as a catalyst. The oligomeric products are predominantly 2′–5′ linked. The chain length and the regioselectivity of the resulting oligoadenylates varies greatly with the concentration of the UO22+ ion catalyst and the pH of the reaction medium. A high total yield of oligouridylates up to dodecamer is also obtained by UO22+ ion catalyst with high 2′–5′ regioselectivity. The oligonucleotide formation is mediated by UO22+-nucleotide complex.

Nucleotide and AP5A complexes of porcine adenylate kinase: a proton and fluorine-19 NMR study

Proton and fluorine nuclear magnetic resonance spectroscopies (NMR) were used as methods to investigate binary complexes between porcine adenylate kinase (AK1) and its substrates. We also studied the interaction of fluorinated substrate analogues and the supposed bisubstrate analogue P1,P5-bis(5'-adenosyl) pentaphosphate (AP5A) with AK1 in the presence of Mg2+. The chemical shifts of the C8-H, C2-H, and ribose C1'-H resonances of both adenosine units in stoichiometric complexes of AK1 with AP5A in the presence of Mg2+ could be determined. The C2-H resonance of one of the adenine bases experiences a downfield shift of about 0.8 ppm on binding to the enzyme. The chemical shift of the His36 imidazole C2-H was changed in the downfield direction on ATP-Mg2+ and, to a lesser extent, AMP binding. 19F NMR chemical shifts of 9-(3-fluoro-3-deoxy-beta-D-xylofuranosyl)adenine triphosphate (3'-F-X-ATP)-Mg2+ and 9-(3-fluoro-3-deoxy-beta-D-xylofuranosyl)adenine monophosphate (3'-F-X-AMP) bound to porcine adenylate kinase could be determined. The different chemical shifts of the bound nucleotides suggest that their mode of binding is different. Free and bound 3'-F-X-AMP are in fast exchange with respect to their 19F chemical shifts, whereas free and bound 3'-F-X-ATP are in slow exchange on the NMR time scale in the absence as well as in the presence of Mg2+. This information could be used to determine the apparent dissociation constants of the nucleotides and the 3'-F-X analogues in the binary complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional characterization of skeletal F‐actin labeled on the NH2‐terminal segment of residues 1–28

Rabbit skeletal alpha-actin was covalently labeled in the filamentous state by the fluorescent nucleophile, N-(5-sulfo-1-naphthyl)ethylenediamine (EDANS) in the presence of the carboxyl group activator 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide (EDC). The coupling reaction was continued until the incorporation of nearly 1 mol EDANS/mol actin. After limited proteolytic digestion of the labeled protein and chromatographic identification of the EDANS-peptides, about 80% of the attached fluorophore was found on the actin segment of residues 1-28, most probably within the N-terminal acidic region of residues 1-7. A minor labeling site was located on the segment that consists of residues 40-113. No label was incorporated into the COOH-terminal moiety consisting of residues 113-375. The isolated EDANS-G-actin undergoes polymerization in the presence of salts but at a rate significantly greater than unlabeled actin. The EDANS-F-actin could be complexed to skeletal chymotryptic myosin subfragment 1 (S-1) and to tropomyosin. The complex formed between EDANS-F-actin and S-1 could not be further crosslinked by EDC but the two proteins were readily joined by glutaraldehyde as observed for native actin-S-1, suggesting that the EDANS-substituted carboxyl site is also involved in the EDC crosslinking of native actin to S-1. Moreover, the EDANS labeling of F-actin resulted in a 20-fold increase in the Km of the actin-activated Mg2+.ATPase of S-1. Thus, this labeling, while it did not much affect the rigor actin-S-1 interaction, changes the actin binding to the S-1-nucleotide complexes significantly. The selective introduction of a variety of spectral probes, like EDANS, or other classes of fluorophores, on the N-terminal region of actin, through the reported carbodiimide coupling reaction, would provide several different derivatives valuable for assessing the functional role of the negatively charged N-terminus of actin during its interaction with myosin and other actin-binding proteins.

Transfer of polarization in the rotating frame. Application to drug—nucleotide complexes

AbstractTransfer of polarization experiments in the rotating frame are used to assign proton resonances in drug‐nucleotide complexes. Transfer by an incoherent process (CAMELSPIN, ROESY) is used to separate contributions from chemical exchange and dipolar interactions, and transfer by a coherent process (HOHAHA, TOCSY) is used to assign scalar coupled protons when lines are broad.

Interaction of Guanine Nucleotides with Ribonuclease F1′ as Studied by Absorption and Circular Dichroism Spectroscopies

Abstract Difference spectra have been observed in both absorption and circular dichroism upon binding of various guanine nucleotides to ribonuclease F1′. The dissociation constants of the enzyrne–nucleotide complexes have been determined based on the difference absorption spectra. The results have been compared with those obtained with ribonuclease T1.

Oxygen toxicity. The influence of adenine-nucleotides and phosphate on Fe2+ autoxidation.

FeCl2 in Na phosphate buffer autoxidizes forming active oxygen species which damage deoxyribose. Di- and triphosphate adenine-nucleotides inhibit both Fe2+ autoxidation and deoxyribose damage in Na phosphate buffer pH 7.4. The inhibition is related to the number of charges of the adenine-nucleotide molecule: ATP at pH 7.4 is a better inhibitor than ADP; at a pH (6.5) close to the pK's of the third and fourth charge of ADP and ATP, ADP inhibition is greatly decreased whereas ATP inhibition is slightly affected. The extent of ATP inhibition of Fe2+ autoxidation depends both on ATP/Mg2+ and ATP/Fe2+ ratios in the reaction mixture. Formation of a Fe2+-nucleotide complex appears to be the mechanism through which ATP and ADP inhibit autoxidation and thus the generation of active oxygen species. These findings are discussed in relation to physiological and pathological fluctuations of nucleotide concentrations.

Production of a streptomycin-Park nucleotide complex by Streptomyces griseus

A compound (compound X) with antibacterial activity was isolated from early-exponential-phase cultures of the streptomycin producer Streptomyces griseus and from protoplast cultures of the same strain. The protoplast cultures produced a larger amount of compound X than did the young hyphae. Both the mycelia and the protoplasts incorporated 14C-labeled myo-inositol, a precursor of streptomycin, into compound X, which has an amino acid content related to that of the cell wall peptidoglycan of the producer strain. Compound X may contain a streptomycin molecule covalently bound to a cell wall precursor unit, i.e., to a Park nucleotide (J. T. Park, J. Biol. Chem. 194:897-904, 1952), through the glutamic acid of the pentapeptide component.

Affinity labeling of the ATP-binding site of type II calmodulin-dependent protein kinase by 5′- p-fluorosulfonylbenzoyl adenosine

Modification of the type II calmodulin-dependent protein kinase by 5′- p-fluorosulfonylbenzoyl adenosine (FSBA) resulted in a time-dependent inactivation of the enzyme. The reaction followed pseudo-first-order kinetics and showed a nonlinear dependence on reagent concentration. The rate of inactivation was sensitive to Mg 2+- and calmodulin-induced conformational changes on the enzyme. However, the enhancing effects of these ligands were not additive; indeed, the kinetic parameters of the Mg 2+-stimulated inactivation reaction with FSBA ( K inact = 2.4 mM; k max = 0.12 min −1) were almost unaffected by the simultaneous addition of calmodulin ( K inact = 1.5 mM; k max = 0.086 min −1). Protection from inactivation by FSBA was provided by Mg 2+-ADP which is consistent with modification of the catalytic site. An analysis of the protective effect of Mg 2+-ADP in the absence ( K d = 590 μM) and presence ( K d = 68 μM) of calmodulin demonstrated that binding of the modulator protein to the enzyme increases the affinity of the protein kinase for nucleotides. Modification by FSBA resulted in labeling of both Tyr and Lys residues but only labeling of Lys was decreased by Mg 2+-ADP which is consistent with the hypothesis that a conserved Lys residue is important in nucleotide binding to the protein kinase. However, the kinetic results of the inactivation reaction suggest that this Lys is not involved in mediating the calmodulin-promoted increase in the affinity of the enzyme for Mg 2+-nucleotide complexes.

Chromium(III) beta, gamma-bidentate guanine nucleotide complexes as probes of the GTP-activated cGMP cascade of retinal rod outer segments.

The exchange-inert Cr(III) beta, gamma-bidentate guanine nucleotide complexes Cr(III)GTP and Cr(III)Gpp(NH)p were used to probe the role of transducin in activating the retinal cGMP cascade. The Cr(III) nucleotide complexes were found to have lower binding affinity for transducin as compared to the Mg2+ complexes. However, the rate of hydrolysis of the transducin-bound Cr(III)GTP was similar to that of Mg(II)GTP. Cr(III)Gpp(NH)p activated the cGMP phosphodiesterase of photolyzed rod outer segment membranes up to 75% of the Mg(II)Gpp(NH)p level but lacked the ability to dissociated the transducin subunits from the rod outer segment membrane. This result implies that the activation of the phosphodiesterase by transducin-GTP complex is a membrane-associated event and the formation of a soluble complex of transducin-GTP with the inhibitory peptide of the phosphodiesterase may not be an obligatory step. Both the delta and lambda screw sense stereoisomers of Cr(III)Gpp(NH)p were capable of activating the cGMP cascade with no apparent stereoselectivity. The nature of the interaction of the metal ion and GTP at the nucleotide-binding site of transducin is discussed together with the results from previous studies using the phosphorothioate GTP analogues [Yamanaka, G., Eckstein, F., & Stryer, L. (1985) Biochemistry 24, 8094-8101] and is compared to the site found in homologous GTP-binding proteins such as elongation factor Tu [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36; la Cour, T.F.M., Nyborg, J., Thirup, S., & Clark, B.F.C. (1985) EMBO J. 4, 2385-2388]. The implications of the observed results on the molecular mechanism of visual signal transduction are discussed.

Effects on the Activity of the Enzyme Phosphoribosyl-Aminoimidazole Carboxylase, Involved in the Biosynthesis of Purine Nucleotides De Novo by Bi-Valent Metal Complexes of the Natural Substrate 5-Amino-1-β-D-Ribofuranosylimidazole-4-Carboxylic Acid 5′-Phosphate

Abstract A series of bi-valent metal complexes of 5-amino-l-β-D-ribofuranosylimida-zole-4-carboxylic acid and its 5′-phosphate derivative (CAIR), a central intermediate in de novo biosynthesis of purine nucleotides have been synthesised. The nucleotide complexes were found to affect the activity of the enzyme phosphoribosylaminoimidazole carboxylase (EC. 4.1.1.21).

Influence of ligands on the aggregation of the normal and mutant forms of phosphofructokinase 2 of Escherichia coli

The aggregation states of Escherichia coli phosphofructokinase 2 (Pfk-2) and of a mutant enzyme (Pfk-2 ∗) altered in the inhibitory allosteric site for MgATP were measured in the presence and in the absence of substrates and products of the reaction. When sucrose gradient ultracentrifugation experiments were performed in the absence of added ligands, both enzymes sedimented as dimers. Likewise, at low concentrations of both substrates (0.1 m m) the aggregation state of Pfk-2 and Pfk-2 ∗ corresponded to a dimer. However, in the presence of 1 m m MgATP alone, Pfk-2 sedimented as a tetramer, whereas Pfk-2 ∗ sedimented as a dimer. At a low fructose 6-phosphate concentration (0.1 m m) and an inhibitory concentration of MgATP (4 m m), Pfk-2 sedimented as a tetramer. However, at the same MgATP concentration but at a higher fructose-6-P concentration (1 m m), a condition under which Pfk-2 is not inhibited by the Mg-nucleotide complex, the enzyme sedimented as a dimer. Pfk-2 ∗ is not inhibited under these conditions and sedimented as a dimer in each case. Thus, the effectiveness of MgATP in promoting the aggregation of Pfk-2 and Pfk-2 ∗ parallels the inhibitability of the enzymes by the nucleotide complex. However, ATP 4−, a potent inhibitor of Pfk-2 and Pfk-2 ∗ that binds to the catalytic site of the enzymes, had no effect upon their aggregation states. Possibly Pfk-2 ∗ is not able to form a tetramer because of an alteration in the regulatory site for the Mg-nucleotide complex.

Biochemical properties of Ha-ras encoded p21 mutants and mechanism of the autophosphorylation reaction.

Kinetic studies performed on p21H guanine nucleotide complexes with and without Mg2+ show that point mutations at positions 12, 59, and 61 each have a different effect on the rate of nucleotide dissociation. Double mutants with a combination of these amino acid substitutions reveal that the effects of each mutation on these kinetics are interactive (nonadditive) for positions 12 and 59 and approximately additive for the positions 12 and 61. The magnitude and direction of the effects seen are dependent on the nature of the nucleotide and whether or not the complexes contain Mg2+. All the mutants have reduced GTPase activity. It is also shown that the autophosphorylation reaction velocity is of first order with respect to the protein concentration and that this reaction is an intramolecular one, which takes place as a side reaction of the GTPase reaction. The autophosphorylation is not reversible under the experimental conditions. The covalently bound phosphate does not decrease the nucleotide-binding ability of the protein nor does it change the relative affinity of the protein for GTP versus GDP. The results are discussed in terms of the structural model and function of p21H.