Synthesis and properties of diastereoisomers of adenosine 5'-(O-1-thiotriphosphate) and adenosine 5'-(O-2-thiotriphosphate).

The chemical synthesis of adenosine 5'-(O-1-thiotriphosphate) (ATPalphaS) and adenosine 5'-(O-2-thiotriphosphate) (ATPbetaS) is described. Both exist as a pair of diastereomers, A and B. The isomers of ATPalphaS can be distinguished on the basis of their different reaction rates with myokinase as well as nucleoside diphosphate kinase. With both enzymes, isomer A reacts fast whereas isomer B reacts considerably more slowly. Phosphorylation of a mixture of isomers of ADPalphaS with pyruvate or acetate kinase yields ATPalphaS, isomer A, whereas the phosphoryl transfer with creatine or arginine kinase yields isomer B. The isomers of ATPbetaS differ in their reactivity with myosin. Isomer A is readily hydrolyzed, whereas isomer B is not. However, isomer B reacts faster with nucleoside diphosphate kinase and ADP than isomer A. Phosphoryl transfer with pyruvate kinase onto ADPbetaS yields ATPbetaS, isomer A, with acetate kinase, isomer B.     

Ionization state of pyridoxal 5'-phosphate in D-serine dehydratase, dialkylglycine decarboxylase and tyrosine phenol-lyase and the influence of monovalent cations as inferred by 31P NMR spectroscopy

The 31P NMR spectroscopy of three pyridoxal 5'-phosphate-dependent enzymes, monomeric D-serine dehydratase, tetrameric dialkylglycine decarboxylase and tetrameric tyrosine phenol-lyase, whose enzymatic activities are dependent on alkali metal ions, was studied. 31P NMR spectra of the latter two enzymes have never been reported, their 3D-structures, however, are available. The cofactor phosphate chemical shift of all three enzymes changes by approximately 3 ppm as a function of pH, indicating that the phosphate group changes from being monoanionic at low pH to dianionic at high pH. The 31P NMR signal of the phosphate group of pyridoxal 5'-phosphate provides a measure of the active site changes that occur when various alkali metal ions are bound. Structural information is used to assist in the interpretation of the chemical shift changes observed. For D-serine dehydratase, no structural data are available but nevertheless the metal ion arrangement in the PLP binding site can be predicted from 31P NMR data.     

Divalent cation-dependent stereospecificity of adenosine 5'-O-(2-thiotriphosphate) in the hexokinase and pyruvate kinase reactions. The absolute stereochemistry of the diastereoisomers of adenosine 5'-O-(2-thiotriphosphate)

31P NMR studies with Cd(II) and Zn(II) chelates of adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) and the Cd(II) chelate of adenosine 5'-O-(2-thiotriphosphate) (ATPbetaS) indicate that these metal ions chelate to the sulfur atom of the thiophosphate group. Since Mg(II) chelates to oxygen of the thiophosphate group of diastereoisomer is equivalent to the configuration of the Cd(II) chelate of the opposite diastereoisomer. As a consequence, an inversion of the stereospecificity is observed when Cd(II) is substituted for Mg(II) in the phosphoryl transfer reactions catalyzed by yeast hexokinase and rabbit muscle pyruvate kinase. When Co(II) is the activating ion for yeast hexokinase with ATPbetaS as substrate, no stereospecificity is observed. Since the absolute configuration for the diastereoisomer of Co(III)(NH3)4ATP which is the active substrate for yeast hexokinase has been established by Cornelius and Cleland (Cornelius, R. D., and Cleland, W. W. (1978) Biochemistry, in press), the absolute stereochemistry of the Mg(II) complex of the B isomer of ATPbetaS is now established by its stereospecificity in the hexokinase reaction.     

Competition between Li+ and Mg2+ for ATP and ADP in aqueous solution: a multinuclear NMR study

We used 7Li NMR spin-lattice relaxation times and 31P NMR chemical shifts to study the binding of Li+ and Mg2+ to the phosphate moieties of ATP and ADP. To examine the binding of Li+ and Mg2+ to the base and ribose moieties, we used 1H and 13C NMR chemical shifts. The 7Li NMR relaxation times of Li+/Mg2+ mixtures of ATP or ADP increased with increasing concentrations of Mg2+, suggesting competition between the two ions for adenine nucleotides. No significant binding of Li+ and Mg2+ to the base and ribose moieties occurred. At the pH and ionic strength used, 2:1 and 1:1 species of the Li(+)-ATP and Li+-ADP complexes were present, with the 2:1 species predominating. In contrast, 1:1 species predominated for the Mg(2+)-ADP and Mg(2+)-ATP complexes. We calculated the Li(+)-nucleotide binding constants in the presence and absence of Mg2+ and found them to be somewhat greater in the presence of Mg2+. Although competition between Li+ and Mg2+ for ATP and ADP phosphate binding sites in solution is consistent with the 31P chemical shift data, the possibility that the Li+ and Mg2+ form mixed complexes with the phosphate groups of ATP or ADP cannot be ruled out.     

31P NMR studies of enzyme-bound substrate complexes of yeast 3-phosphoglycerate kinase. 1. Effects of sulfate and pH. Mg(II) affinity at the two ATP sites

31P NMR measurements were made (at 121.5 MHz and 5 degrees C) on enzyme-bound substrate complexes of 3-phosphoglycerate kinase in order to address three questions pertaining to (i) the integrity of the enzyme-substrate complexes with Mg(II) in the presence of sulfate concentrations typical of those used for crystallization in X-ray studies, (ii) the relative affinities of Mg(II) to ATP bound at the two sites on the enzyme, and (iii) the pH behavior of the different phosphate groups in the enzyme complexes. 31P chemical shift and spin-spin coupling constant changes showed that at concentrations of 0.5 M and higher, sulfate ion interferes with Mg(II) chelation to ATP and ADP free in solution as well as in their enzyme-bound complexes. The effect on enzyme complexes is stronger for the E.MgATP complex than for the E.MgADP complex. Sulfate ion (50 mM) also causes a approximately 0.5 ppm upfield chemical shift of the 31P resonance of enzyme-bound 3-P-glycerate even in the absence of ATP or Mg(II). A quantitative estimate of the dispartate affinities of Mg(II) to ATP bound at the two sites on the enzyme was made on the basis of computer simulation of changes in the line shape of beta-P (ATP) resonance and of changes in 31P chemical shift of the corresponding gamma-P (ATP) in the E.ATP complex with increasing [Mg(II)]. The concentrations of the relevant species that contribute to these 31P NMR signals were computed by assuming independent binding at the two sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metal-phosphate interactions in the hammerhead ribozyme observed by 31P NMR and phosphorothioate substitutions

The hammerhead ribozyme is a catalytic RNA that requires divalent metal cations for activity under moderate ionic strength. Two important sites that are proposed to bind metal ions in the hammerhead ribozyme are the A9/G10.1 site, located at the junction between stem II and the conserved core, and the scissile phosphate (P1.1). (31)P NMR spectroscopy in conjunction with phosphorothioate substitutions is used in this study to investigate these putative metal sites. The (31)P NMR feature of a phosphorothioate appears in a unique spectral window and can be monitored for changes upon addition of metals. Addition of 1-2 equiv of Cd(2+) to the hammerhead with an A9-S(Rp) or A9-S(S)(Rp) substitution results in a 2-3 ppm upfield shift of the (31)P NMR resonance. In contrast, the P1.1-S(Rp) and P1.1-S(Sp) (31)P NMR features shift slightly and in opposite directions, with a total change in delta of 相似文献   

Phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate: 31P nuclear magnetic resonance study

31P nuclear magnetic resonance (NMR) has been used to study the 1-phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate (P-Rib-PP). Comparison of the proton-decoupled spectra of 5-phosphoribosyl 1-O-(2-thiodiphosphate) (P-Rib-PP beta S) and the SP diastereomer of 5-phosphoribosyl 1-O-(1-thiodiphosphate) (P-Rib-PP alpha S) with the parent molecule revealed a characteristic large downfield chemical shift change for the resonance signal associated with the thiophosphate group (delta delta approximately 40-50 ppm) and an increase in the magnitude of the phosphate-thiophosphate spin-spin coupling constant (delta J alpha beta approximately 10 Hz). Both these changes are consistent with the observed effects of sulfur substitution on the behavior of the adenosine nucleotides, particularly ADP [Jaffe, E. K., & Cohn, M. (1978) Biochemistry 17, 652-657]. High-field 31P NMR has also been used to demonstrate the diastereomeric purity of P-Rib-PP alpha S (Sp diastereomer) and the greater lability of this analogue when compared with both P-Rib-PP beta S and P-Rib-PP. Sulfur substitution was found to cause a large decrease in the apparent pKa associated with the thiophosphate moiety of P-Rib-PP beta S (delta pKa approximately 1.4 units) and also to enhance the sensitivity of the thiophosphate chemical shift to protonation and, in particular, to Mg2+ binding, compared with P-Rib-PP. The potential application of the phosphorothioate analogues as probes of the reactions catalyzed by the phosphoribosyltransferase enzymes is discussed.     

Equilibrium study on the interaction of phytic acid with polyamines and metal ions

Interaction of phytic acid (myo-inositolhexakisphosphoric acid, IP) and polyamines (A = en, tn, Put, dien, 2,3-tri, 3,3-tri, Spd, 3,3,3-tet, spermine(Spm)) have been studied by potentiometric and (31)P-NMR techniques. The non-covalent interactions have led to the formation of stable molecular complexes of (IP)H(n)(A) type at the 1:1 molar ratio of the ligands, but of different numbers of protons. The IP protonation constants, stability constants of the molecular complexes and metal (Mg(2+)) complexes have been determined. The structural and pH dependences of stability constants showed the interactions between IP and A have the acid-base character determining their effectiveness, although the IP structure (5ax1eq, 5eq1ax) in molecular complexes should be also taken into account. (31)P NMR study showed in the presence of Spm (31)P highfield shifts and high pH shift of signal broadening due to chemical exchange between 5ax1eq and 5eq1ax. The preferable binding of Spm to IP over Mg(2+) in neutral pH indicated the importance of polyamine as a stabilizer of phosphate compounds.     

Ribozyme-catalyzed and nonenzymatic reactions of phosphate diesters: rate effects upon substitution of sulfur for a nonbridging phosphoryl oxygen atom

The L-21 ScaI ribozyme derived from the intervening sequence of Tetrahymena thermophila pre-rRNA catalyzes a guanosine-dependent endonuclease reaction that is analogous to the first step in self-splicing of this intervening sequence. We now describe pre-steady-state kinetic experiments, with sulfur substituting for the pro-RP (nonbridging) phosphoryl oxygen atom at the site of cleavage, that test aspects of a kinetic model proposed for the ribozyme reaction (Herschlag, D., & Cech, T. R. (1990) Biochemistry 29, 10159-10171). Thio substitution does not affect the reaction with subsaturating oligonucleotide substrate and saturating guanosine ((kcat/Km)S), consistent with the previous finding that binding of the oligonucleotide substrate limits this rate constant. In contrast, there is a significant decrease in the rate of single-turnover reactions of ribozyme-bound (i.e., saturating) oligonucleotide substrate upon thio substitution, with decreases of 2.3-fold for the reaction with guanosine ((kcat/Km)G) and 7-fold for hydrolysis [i.e., with solvent replacing guanosine; kc(-G)]. These "thio effects" are consistent with rate-limiting chemistry, as shown by comparison with model reactions. Nonenzymatic nucleophilic substitution reactions of the phosphate diester, methyl 2,4-dinitrophenyl phosphate monoanion, are slowed 4-11-fold by thio substitution for reactions with hydroxide ion, formate ion, fluoride ion, pyridine, and nicotinamide. In addition, we have confirmed that thio substitution has no effect on the nonenzymatic alkaline cleavage of RNA (Burgers, P. M. J., & Eckstein, F. (1979) Biochemistry 18, 592-596). Considering the strong preference of Mg2+ for binding to oxygen rather than sulfur, the modest thio effect on the chemical step of the ribozyme-catalyzed reaction and the absence of a thio effect on the equilibrium constant for binding of the oligonucleotide substrate suggest that the pro-RP oxygen atom is not coordinated to Mg2+ in the E.S complex or in the transition state. General implications of thio effects in enzymatic reactions of phosphate diesters are discussed.     

ATPalphaS is a ligand for P2Y receptors in synaptosomal membranes: solubilization of [35S]ATPalphaS binding proteins associated with G-proteins.

ATPalphaS was established as a P2Y receptor-specific ligand for assaying the solubilization of functional native P2Y receptors from synaptosomal membranes. These receptors are not yet amenable to biochemical studies. High-affinity [35S]ATPalphaS binding sites in synaptosomal membranes, solubilized with Brij58, retained the binding affinity and ligand specificity (ATPalphaS = ATP > 2-MeSATP > ADP, ADPbetaS > AMP > alpha,beta-MeATP) corresponding to P2Y receptors. Mg2+ but not Ca2+, enhanced high-affinity [35S]ATPalphaS binding 30-fold, supporting specific recognition by P2Y receptors. ATPalphaS stimulated P2Y receptor-mediated [35S]GTPgammaS binding equipotently with ATP in synaptosomal membranes and in Brij58-solubilized proteins demonstrating the association with G-proteins. Anion-exchange chromatography of solubilized synaptosomal membrane proteins yielded two fractions in which [35S]ATPalphaS binding was regulated by GTPgammaS/Mg2+, thus possibly by heterotrimeric G-proteins. After a second chromatographic step (hydroxyapatite) the regulation of high-affinity [35S]ATPalphaS binding by Mg2+ was still present, whereas the regulation by GTPgammaS/Mg2+ was lost indicating the dissociation from G-proteins. Thus, conditions were found to stabilize ligand binding activity of solubilized P2Y receptors and to solubilize P2Y receptors associated with G-proteins.     

31P nuclear magnetic resonance study of phosphoribosyldiphosphate and its interaction with magnesium ions

31P NMR has been used to study phosphoribosyldiphosphate (P-Rib-PP) over a wide range of pH values, both in the absence and presence of MgCl2. In the absence of MgCl2, the chemical shift variations of the three 31P nuclei in the molecule, over the pH range 4 to 9, were found to be largest for the terminal 1-diphosphate (1P beta) oxyanion and the 5-phosphate (5P) moiety. Apparent pK alpha values of approximately 6.1 and 6.3 were estimated for protonation of the 1P beta and 5P groups, respectively. Variations in the apparent pK alpha values associated with 1P beta and 5P oxyanions in the presence of various concentrations of MgCl2 were consistent with P-Rib-PP having two independent metal ion binding sites with different affinities for Mg2+ ions. The binding of Mg2+ reduced the apparent pK alpha of the 1P beta moiety by approximately 1.6 units and the apparent pK alpha of the 5P group by approximately 0.7 unit. This behavior is analogous to the situation reported for the terminal phosphooxyanion of ADP and observed for the phosphate group of ribose 5-phosphate, respectively. In the presence of an equimolar concentration of added MgCl2, the 1P alpha and 1P beta resonances of P-Rib-PP were shifted downfield and the 31P-31P coupling constant was decreased. Changes in both these parameters were very similar to those reported for the MgADP- complex. The observed chemical shifts and spin-spin coupling constants suggest that the diphosphate and monophosphate moieties of P-Rib-PP act as independent binding sites for Mg2+ in a manner similar to the phosphooxyanion groups of ADP and ribose 5-phosphate, respectively.     

High-resolution phosphorus nuclear magnetic resonance spectra of yeast phenylalanine transfer ribonucleic acid. Melting curves and relaxation effects.

In a continuation of our studies on structural effects on the 31P chemical shifts of nucleic acids, we present 31P NMR spectra of yeast phenylalanine tRNA in the presence and absence of Mg2+. Superconducting field (146 MHz) and 32-MHz 31P NMR spectra reveal approximately 15 nonhelical diester signals spread over approximately 7 ppm besides the downfield terminal 3'-phosphate monoester. In the presence of 10 mM Mg2+, most scattered and main cluster signals do not shift between 22--66 degrees C, thus supporting our earlier hypothesis that 31P chemical shifts are sensitive to phosphate ester torsional and bond angles. At 70 degrees C, all of the signals merge into a single random coil conformation signal. Similar effects are observed in the absence of Mg2+ except that the transition melting temperature is approximately 20 degrees C lower. Measured spin-lattice and spin-spin relaxation times reveal another lower temperature transition besides the thermal denaturation process. A number of the scattered peaks are shifted (0.2--1.7 ppm) and broadened between 22 and 66 degrees C in the presence of Mg2+ as a result of this conformational transition between two intact tertiary structures. The loss of the scattered peaks in the absence of Mg2+ occurs in the temperature range expected for melting of a tertiary structure. An attempt to simulate the 31P spectra of tRNA Phe based upon the X-ray crystallographically determined phosphate ester torsional agles supports the suggestion that the large shifts in the scattered peaks are due to bond angle distortions in the tertiary structure.     

NMR studies and semi-empirical energy calculations for cyclic ADP-ribose

A possible pH-dependent conformational switch was investigated for cyclic ADP-ribose. NMR signals for the exchangeable protons were observed in H2O at low temperature, but there was no direct evidence for the protonation of N-3 at neutral pH that has previously been postulated. MNDO calculations indicated that pH dependent 31P chemical shift changes are attributable to protonation of the phosphate adjacent to the N-1 of adenine, and not due to trans-annular hydrogen bonding with a protonated N-3.     

Phosphorus-31 Fourier transform nuclear magnetic resonance study of mononucleotides and dinucleotides. 1. Chemical shifts.

A phosphorus-31 nuclear magnetic resonance (NMR) study of adenine, uracil, and thymine mononucleotides, their cyclic analogues, and the corresponding dinucleotides is reported. From the pH dependence of phosphate chemical shifts, pKa values of 6.25-6.30 are found for all 5'-mononucleotides secondary phosphate ionization, independently from the nature of the base and the presence of a hydroxyl group at the 2' position. Conversely, substitution of a hydrogen atom for a 2'-OH lowers the pKa of 3'-monoribonucleotides from 6.25 down to 5.71-5.85. This indication of a strong influence of the 2'-hydroxyl group on the 3'-phosphate is confirmed by the existence of a 0.4 to 0.5 ppm downfield shift induced by the 2'-OH on the phosphate resonance of 3'-monoribonucleotides, and 3',5'-cyclic nucleotides and dinucleotides with respect to the deoxyribosyl analogues. Phosphate chemical shifts and titration curves are affected by the ionization and the type of the base. Typically, deviations from the theoretical Henderson-Hasselbalch plots are observed upon base titration. In addition, purine displays a more deshielding influence than pyrimidine on the phosphate groups of most of the mononucleotides (0.10 to 0.25 ppm downfield shift) with a reverse situation for dinucleotides. These effects together with the importance of stereochemical arrangement (furanose ring pucker, furanose-phosphate backbone conformation, O-P-O bond angle) on the phosphate chemical shifts are discussed.     

Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids.     

(31)P NMR spectroscopy senses the microenvironment of the 5'-phosphate group of enzyme-bound pyridoxal 5'-phosphate

In this review it is demonstrated that (31)P NMR spectroscopy can be used to elucidate information about the microenvironment around the phosphate group of enzyme-bound pyridoxal 5'-phosphate (PLP). The following information can be obtained for all PLP-dependent enzymes: 1) the protonation state of the 5'-phosphate and its exposure to solvent, and 2) tightness of binding of the 5'-phosphate. In addition, the 5-phosphate can report on the protonation state of the Schiff base lysine in some enzymes. Changes in the 5'-phosphate chemical shift can be used to determine changes in tightness of binding of the phosphate as the reaction pathway is traversed, providing information on the dynamics of the enzyme. (31)P NMR spectroscopy is thus an important probe of structure, dynamics and mechanism in native and site-directed mutations of PLP-dependent enzymes. Examples of all of the above are provided in this review. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.     

31P nuclear magnetic resonance spectroscopy studies of substrate and product binding to fructose-1,6-bisphosphatase.

The enzymatic hydrolysis of fructose 1,6-bisphosphate (Fru-1,6-P2) to fructose 6-phosphate (Fru-6-P) and inorganic phosphate (Pi), which is catalyzed by fructose-1,6-bisphosphatase, has been studied by 31P nuclear magnetic resonance spectroscopy (NMR). At pH 7.5 and 15 degrees C, the equilibrium constant for the central complex K'eq = [E.Fru-6-P.Pi]/[E.Fru-1,6-P2.H2O] is about 2. This observation is in harmony with results obtained with a number of Bi Bi enzyme systems for the determination of K'eq in which a variety of experimental techniques were used (Knowles, J.R. (1980) Annu. Rev. Biochem. 49, 877-919). Significant changes in 31P NMR chemical shifts were observed for both the substrate, Fru-1,6-P2, and the product, Fru-6-P, when bound to the enzyme relative to ligand free in solution. The chemical shifts of the substrate and product were altered further in the presence of Mg2+, the catalytic divalent metal ion. The chemical shifts caused by the addition of metal ion can be reversed in the presence of trans-1,2-diaminocyclohexane- N,N,N',N'-tetraacetic acid (CDTA) or AMP. In the presence of the metal ion chelator or the nucleotide, the substrate had a chemical shift that was about the same as that observed in the absence of metal ion. On the basis of these observations we suggest that AMP and CDTA exhibit similar effects, i.e. they both remove the catalytic metal ion from the enzyme. This finding is supportive of the suggestion (Scheffler, J. E., and Fromm, H.J. (1986) Biochemistry 25, 6659-6665; Liu, F., and Fromm, H.J. (1990) J. Biol. Chem. 265, 7401-7406) that the role of AMP in the regulation of fructose-1,6-bisphosphatase is to prevent binding of the divalent metal activator to the enzyme.     

Nucleotide and AP5A complexes of porcine adenylate kinase: A 1H and 19F 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)     

Phosphorus-31 nuclear magnetic resonance studies of the binding of nucleotides to NADP+-specific isocitrate dehydrogenase

The interaction of the 2'-phosphate-containing nucleotides (NADP+, NADPH, 2'-phosphoadenosine 5'-diphosphoribose, and adenosine 2',5'-bisphosphate) with NADP+ -specific isocitrate dehydrogenase was studied by using 31P NMR spectroscopy. The separate resonances corresponding to free and bound nucleotides, characteristic for slow exchange of nuclei on the NMR time scale, were observed in the spectra of the enzyme (obtained in the presence of excess ligand) with NADP+ and NADPH in the absence and presence of Mg2+ and with 2'-phosphoadenosine 5'-diphosphoribose in the absence of metal or in the presence of the substrate magnesium isocitrate. The position of the 31P resonance of the bound 2'-phosphate group in these spectra is invariant (delta = 6) in the pH range 5-8, indicating that the pK of this group is much lower in the complexes with the enzyme than that (pK = 6.13) in the free nucleotides. The additional downfield shift of this resonance by 1.8 ppm beyond that (delta = 4.22) of the dianionic form of the 2'-phosphate in free nucleotides suggests interaction with a positively charged group(s) and/or distortion of P-O-P angles as the result of binding to the enzyme. A single resonance of 2'-phosphate was observed in the spectrum of the enzyme complex with 2'-phosphoadenosine 5'-diphosphoribose in the presence of Mg2+, with the chemical shift dependent on the nucleotide to enzyme ratio, characteristic for the fast exchange situation. Addition of metal does not perturb the environment of the 2'-phosphate in the complexes of NADP+ and NADPH with isocitrate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

A phosphorus-magnetic-resonance study of the interaction of Mg2+ with adenyl-5'-yl imidodiphosphate. Binding sites of Mg2+ ion on the phosphate chain.

The interaction of Mg2+ ions with adenyl-5'-yl imidodiphosphate, AMP-P(NH)P, has been studied at basic and acidic pH values by phosphorus magnetic resonance spectroscopy in aqueous solution. The results suggest that Mg2+ binds simultaneously to one (or both) of the two free oxygen atoms of the beta-phosphate moiety and to the nitrogen atom of the phosphate chain (P alpha-O-P beta-N-P gamma). The interaction arises from 1: 1 complexing of Mg2+ to AMP-P(NH)P. The mode of the Mg2+ binding on the phosphate chain remains the same at both basic and acidic pH values. As in the case of ATP and ADP, the association of Mg2+ reduces the pK by about 1.5 units. On the other hand phosphorus titration curves showed that when the phosphate chain does not possess the regular periodicity (O-P alpha-O-P beta-X-P gamma-O,X not equal to O) as in the case of ATP, protonation of the terminal phosphate group may induce a 31P chemical shift variation less important for this group than for the preceding one.