Enzyme control of small-molecule coordination in FosA as revealed by 31P pulsed ENDOR and ESE-EPR

FosA is a manganese metalloglutathione transferase that confers resistance to the broad-spectrum antibiotic fosfomycin, which contains a phosphonate group. The active site of this enzyme consists of a high-spin Mn(2+) ion coordinated by endogenous ligands (a glutamate and two histidine residues) and by exogenous ligands, such as substrate fosfomycin. To study the Mn(2+) coordination environment of FosA in the presence of substrate and the inhibitors phosphonoformate and phosphate, we have used (31)P pulsed electron-nuclear double resonance (ENDOR) at 35 GHz to obtain metrical information from (31)P-Mn(2+) interactions. We have found that continuous wave (CW) (31)P ENDOR is not successful in the study of phosphates and phosphonates coordinated to Mn(2+). Parallel studies of phosph(on)ate binding to the Mn(2+) of FosA and to aqueous Mn(2+) ion disclose how the enzyme modifies the coordination of these molecules to the active site Mn(2+). Through analysis of (31)P hyperfine parameters derived from simulations of the ENDOR spectra we have determined the binding modes of the phosph(on)ates in each sample and discerned details of the geometric and electronic structure of the metal center. The (31)P ENDOR studies of the protein samples agree with, or improve on, the Mn-P distances determined from crystal structures and provide Mn-phosph(on)ate bonding information not available from these studies. Electron spin echo electron paramagnetic resonance (ESE-EPR) spectra have also been recorded. Simulation of these spectra yield the axial and rhombic components of the Mn(2+) (S = (5)/(2)) zero-field splitting (zfs) tensor. Comparison of structural inferences based on these zfs parameters both with the known enzyme structures and the (31)P ENDOR results establishes that the time-honored procedure of analyzing Mn(2+) zfs parameters to describe the coordination environment of the metal ion is not valid or productive.     

Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate.

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, KD = 1-2 muM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (KI = 240-300 muM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This "tightly bound" ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetics studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, Pi can maintain the active form of the enzyme.     

The Mn(2+)-bicarbonate complex in a frozen solution revisited by pulse W-band ENDOR

The coordination of bicarbonate to Mn (2+) is the simplest model system for the coordination of Mn (2+) to carboxylate residues in a protein. Recently, the structure of such a complex has been investigated by means of X-band pulse EPR (electron paramagnetic resonance) experiments ( Dasgupta, J. ; et al. J. Phys. Chem. B 2006, 110, 5099 ). Based on the EPR results, together with electrochemical titrations, it has been concluded that the Mn (2+) bicarbonate complex consists of two bicarbonate ligands, one of which is monodentate and other bidentate, but only the latter has been observed by the pulsed EPR techniques. The X-band measurements, however, suffer several drawbacks. (i) The zero-field splitting (ZFS) term of the spin Hamiltonian affects the nuclear frequencies. (ii) There are significant contributions from ENDOR (electron nuclear double resonance) lines of the M S not equal +/- (1)/ 2 manifolds. (iii) There are overlapping signals of (23)Na. All these reduce the uniqueness of the data interpretation. Here we present a high-field ENDOR investigation of Mn (2+)/NaH (13)CO 3 in a water/methanol solution that eliminates the above difficulties. Both Davies and Mims ENDOR measurements were carried out. The spectra show that a couple of slightly inequivalent (13)C nuclei are present, with isotropic and anisotropic hyperfine couplings of A iso1 = 1.2 MHz, T perpendicular1 = 0.7 MHz, A iso2 = 1.0 MHz, T perpendicular2 = 0.6 MHz, respectively. The sign of the hyperfine coupling was determined by variable mixing time (VMT) ENDOR measurements. These rather close hyperfine parameters suggest that there are either two distinct, slightly different, carbonate ligands or that there is some distribution in conformation in only one ligand. The distances extracted from T perpendicular1 and T perpendicular2 are consistent with a monodentate binding mode. The monodentate binding mode and the presence of two ligands were further supported by DFT calculations and (1)H ENDOR measurements. Additionally, (23)Na ENDOR resolved at least two types of (23)Na (+) in the Mn (2+)-bicarbonate complex, thus suggesting that the bicarbonate bridges two positively charged metal ions.     

Metal ion induced allosteric transition in the catalytic activity of an artificial phosphodiesterase

An artificial phosphodiesterase () bearing two types of metal binding sites, a catalytic site and a regulatory bipyridine site showed a unique allosteric transition in the catalytic activity against the metal concentration. The rate constants for the hydrolysis reaction of 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) and RNA dimer (ApA) with and without an effector metal ion were evaluated; the k(obs) value of HPNP hydrolysis for .(Zn(2+))(3) (2.0 x 10(-4) s(-1)) is 3.3 times larger than that for .(Zn(2+))(2). In the case of and Cu(2+), a 19.4 times larger k(obs) value was obtained for .(Cu(2+))(3) (1.2 x 10(-3) s(-1)) against .(Cu(2+))(2). The increase in the catalytic activity is ascribed to the allosteric conformational transition of induced by the coordination of effector metal ion to the Bpy moiety. A detailed investigation revealed that a conformational change of induced by the third M(2+) complexation enhances the rate of hydrolysis rather than a change in the substrate affinity.     

Testing for violations of microscopic reversibility in ATP-sensitive potassium channel gating

In pancreatic beta cells, insulin secretion is tightly controlled by the cells' metabolic state via the ATP-sensitive potassium (KATP) channel. ATP is a key mediator in this signaling process, where its role as an inhibitor of KATP channels has been extensively studied. Since the channel contains an ATPase as an accessory subunit, the possibility that ATP hydrolysis mediates KATP channel opening has also been proposed. However, a rigorous test of coupling between ATP hydrolysis and channel gating has not previously been performed. In the present work, we examine whether KATP channel gating obeys detailed balance in order to determine whether ATP hydrolysis is strongly coupled to the gating of the KATP channel. Single-channel records were obtained from inside-out patches of transiently transfected HEK-293 cells. Channel activity in membrane patches with exactly one channel shows no violations of microscopic reversibility. Although KATP channel gating shows long closed times on the time scale where ATP hydrolysis takes place, the time symmetry of channel gating indicates that it is not tightly coupled to ATP hydrolysis. This lack of coupling suggests that channel gating operates close to equilibrium; although detailed balance is not expected to hold for ATP hydrolysis, it still does so in channel gating. On the basis of these results, the function of the ATPase active site in channel gating may be to sense nucleotides by differential binding of ATP and ADP, rather than to drive a thermodynamically unfavorable conformational change.     

Exploring sites on mitochondrial ATPase for catalysis, regulation, and inhibition.

Evidence is presented that mitochondrial ATPase has two types of sites that bind adenine nucleotides. The catalytic site, C, binds the substrates ATP, GTP, or ITP and the inhibitor guanylyl imidodiphosphate (GMP-PNP). A second type of site, R, binds ATP, ADP, adenylyl imidodiphosphate (AMP-PNP), and the chromium complexes of ATP or ADP. All of these substances binding to the R site inhibit the hydrolysis of ATP in a competitive manner; their inhibition of hydrolysis of ITP and GTP is noncompetitive. GMP-PNP inhibits oxidative phosphorylation in submitochondrial particles but AMP-PNP does not. The localization on mitochondrial membranes of sites for the binding of various antibiotics that inhibit oxidative phosphorylation is discussed.     

Magnetic resonance and kinetic studies of the mechanism of membrane-bound sodium and potassium ion- activated adenosine triphosphatase.

EPR and water proton relaxation rate (1/T1) studies of partially (40%) and "fully" (90%) purified preparations of membrane-bound (Na+ + K+) activated ATPase from sheep kidney indicate one tight binding site for Mn2+ per enzyme dimer, with a dissociation constant (KD = 0.88 muM) in agreement with the kinetically determined activator constant, identifying this Mn2+-binding site as the active site of the ATPase. Competition studies indicate that Mg2+ binds at this site with a dissociation constant of 1 mM in agreement with its activator constant. Inorganic phosphate and methylphosphonate bind to the enzyme-Mn2+ complex with similar high affinities and decrease 1/T1 of water protons due to a decrease from four to three in the number of rapidly exchanging water protons in the coordination sphere of enzyme-bound Mn2+. The relative effectiveness of Na+ and K+ in facilitating ternary complex formation with HPO2-4 and CH3PO2-3 as a function of pH indicates that Na+ induces the phosphate monoanion to interact with enzyme-bound Mn2+. Thus protonation of an enzyme-bound phosphoryl group would convert a K+-binding site to a Na+-binding site. Dissociation constants for K+ and Na+, estimated from NMR titrations, agreed with kinetically determined activator constants of these ions consistent with binding to the active site. Parallel 32Pi-binding studies show negligible formation (less than 7%) of a covalent E-P complex under these conditions, indicating that the NMR method has detected an additional noncovalent intermediate in ion transport. Ouabain, which increases the extent of phosphorylation of the enzyme to 24% at pH 7.8 and to 106% at pH 6.1, produced further decreases in 1/T1 of water protons. Preliminary 31P- relaxation studies of CH3PO2-3 in the presence of ATPase and Mn2+ yield an Mn to P distance (6.9 +/- 0.5 A) suggesting a second sphere enzyme-Mn-ligand-CH3PO2-3 complex. Previous kinetic studies have shown that T1+ substitutes for K+ in the activation of the enzyme but competes with Na+ at higher levels. From the paramagnetic effect of Mn2+ at the active site on the enzyme on I/T1 of 205T1 bound at the Na+ site, a Mn2+ to T1+ distance of 4.0 +/- 0.1 A is calculated, suggesting the sharing of a common ligand atomy by Mn2+ and T1+ on the ATPase. Addition of Pi increases this distance to 5.4 A consistent with the insertion of P between Mn2+ and T1+. These results are consistent with a mechanism for the (Na+ + K+)-ATPase and for ion transport in which the ionization state of Pi at a single enzyme active site controls the binding and transport of Na+ and K+, and indicate that the transport site for monovalent cations is very near the catalytic site of the ATPase. Our mechanism also accounts for the order of magnitude weaker binding of Na+ compared to K+.     

Probing the ATP hydrolysis cycle of the ABC multidrug transporter LmrA by pulsed EPR spectroscopy

Members of the ATP binding cassette (ABC) transporter superfamily translocate various types of molecules across the membrane at the expense of ATP. This requires cycling through a number of catalytic states. Here, we report conformational changes throughout the catalytic cycle of LmrA, a homodimeric multidrug ABC transporter from L. lactis. Using site-directed spin labeling and pulsed electron-electron double resonance (PELDOR/DEER) spectroscopy, we have probed the reorientation of the nucleotide binding domains and transmembrane helix 6 which is of particular relevance to drug binding and part of the dimerization interface. Our data show that LmrA samples a very large conformational space in its apo state, which is significantly reduced upon nucleotide binding. ATP binding but not hydrolysis is required to trigger this conformational change, which results in a relatively fixed orientation of both the nucleotide binding domains and transmembrane helices 6. This orientation is maintained throughout the ATP hydrolysis cycle until the protein cycles back to its apo state. Our data present strong evidence that switching between two dynamically and structurally distinct states is required for substrate translocation.     

Artificial Accelerators of the Molecular Chaperone Hsp90 Facilitate Rate‐Limiting Conformational Transitions

The molecular chaperone Hsp90 undergoes an ATP‐driven cycle of conformational changes in which large structural rearrangements precede ATP hydrolysis. Well‐established small‐molecule inhibitors of Hsp90 compete with ATP‐binding. We wondered whether compounds exist that can accelerate the conformational cycle. In a FRET‐based screen reporting on conformational rearrangements in Hsp90 we identified compounds. We elucidated their mode of action and showed that they can overcome the intrinsic inhibition in Hsp90 which prevents these rearrangements. The mode of action is similar to that of the co‐chaperone Aha1 which accelerates the Hsp90 ATPase. However, while the two identified compounds influence conformational changes, they target different aspects of the structural transitions. Also, the binding site determined by NMR spectroscopy is distinct. This study demonstrates that small molecules are capable of triggering specific rate‐limiting transitions in Hsp90 by mechanisms similar to those in protein cofactors.     

Single crystal 55Mn ENDOR of concanavalin a: detection of two Mn2+ sites with different 55Mn quadrupole tensors

Concanavalin A is a member of the plant hemeagglutinin (or plant lectin) family that contains two metal binding sites; one, called S1, is occupied by Mn2+ and the other, S2, by Ca2+. 55Mn electron-nuclear double resonance (ENDOR) measurements were performed on a single crystal of concanavalin A at W-band (95 GHz, ~3.5 T) to determine the 55Mn nuclear quadrupole interaction in a protein binding site and its relation to structural parameters. Such measurements are easier at a high field because of the high sensitivity for size-limited samples and the reduction of second-order effects on the spectrum which simplifies spectral analysis. The analysis of the 55Mn ENDOR rotation patterns showed that two chemically inequivalent Mn2+ types are present at low temperatures, although the high-resolution X-ray structure reported only one site. Their quadrupole coupling constants, e2Qq/h, are significantly different; 10.7 +/- 0.6 MHz for Mand only -2.7 +/-0.6 MHz for M. The ENDOR data also refined the hyperfine coupling determined earlier by single-crystal EPR measurements, yielding a small but significant difference between the two: -262.5 MHz for M and -263.5 MHz for M. The principal z-axis for M is not aligned with any of the Mn-ligand directions, but is 25 off the Mn-asp10 direction, and its orientation is different than that of the zero-field splitting (ZFS) interaction. Because of the small quadrupole interaction of M the orientation dependence was very mild, leading to larger uncertainties in the asymmetry parameter. Nonetheless, there too z is not along the Mn-ligand bonds and is rotated 90 with respect to MnA. These results show, that similar to the ZFS, the quadrupolar interaction is highly sensitive to small differences in the coordination sphere of the Mn2+, and the resolution of the two types is in agreement with the earlier observation of a two-site conformational dynamic detected through the ZFS interaction, which is frozen out at low temperatures and averaged at room temperature. To account for the structural origin of the different e2Qq/h values, the electric field gradient tensor was calculated using the point-charge model. The calculations showed that a relatively small displacement of the oxygen ligand of asp10 can lead to differences on the order observed experimentally.     

ENERGY-REGULATED FUNCTIONAL TRANSITIONS OF CHLOROPLAST ATPASE

The functional transitions of the membrane-bound chloroplast ATPase (CF₁) as influenced by low ADP and uncoupler concentrations are investigated by measurements of initial and steady-state ATP hyrolysis and concomitant membrane energization. Following activation of latent ATP hydrolysis by light in the presence of dithioerythritol, the resulting steady-state ATP hydrolysis depends on the dark-period ( t _d) bteween light activation and ATP addition. ADP, added during t _d, inhibits this activity ( K _i about 2 μ M ) and induces a lag in the onset of ATP hydrolysis. The extent of membrane energization as monitored by an aminoacridine fluorescent probe is proportional to the ATPase activity.
An uncoupler amplifies the inhibitory effect of ADP if added during f _d, whereas it induces the normal stimulation of ATP hydrolysis in the absence of ADP. The ADP effect, which is different from product inhibition, is interpreted as a conformational interaction with CF₁ causing an increase of the energy threshold required for the inactive → active transition of the CF₁ molecules. These results are in harmony with currently proposed models of CF₁ regulation by adenine nucleotides based on binding studies.
The inactive → active transition of CF₁ conformation is investigated by analysis of the lag in the onset of ATP hydrolysis at different ADP concentrations and by means of varied light pulses and single-turnover flashes, using the electric potential indicating absorption change at 515 nm as a probe for the onset of ATP hydrolysis. The half-time of the process leading to fully (re)activated ATP hydrolysis is about 0.25 s. The ATP-dependent flash-induced inactive → active transition occurs within a few turnovers of electron flow.     

Effect of Ca2+/Sr2+ substitution on the electronic structure of the oxygen-evolving complex of photosystem II: a combined multifrequency EPR, 55Mn-ENDOR, and DFT study of the S2 state

The electronic structures of the native Mn(4)O(x)Ca cluster and the biosynthetically substituted Mn(4)O(x)Sr cluster of the oxygen evolving complex (OEC) of photosystem II (PSII) core complexes isolated from Thermosynechococcus elongatus, poised in the S(2) state, were studied by X- and Q-band CW-EPR and by pulsed Q-band (55)Mn-ENDOR spectroscopy. Both wild type and tyrosine D less mutants grown photoautotrophically in either CaCl(2) or SrCl(2) containing media were measured. The obtained CW-EPR spectra of the S(2) state displayed the characteristic, clearly noticeable differences in the hyperfine pattern of the multiline EPR signal [Boussac et al. J. Biol. Chem.2004, 279, 22809-22819]. In sharp contrast, the manganese ((55)Mn) ENDOR spectra of the Ca and Sr forms of the OEC were remarkably similar. Multifrequency simulations of the X- and Q-band CW-EPR and (55)Mn-pulsed ENDOR spectra using the Spin Hamiltonian formalism were performed to investigate this surprising result. It is shown that (i) all four manganese ions contribute to the (55)Mn-ENDOR spectra; (ii) only small changes are seen in the fitted isotropic hyperfine values for the Ca(2+) and Sr(2+) containing OEC, suggesting that there is no change in the overall spin distribution (electronic coupling scheme) upon Ca(2+)/Sr(2+) substitution; (iii) the changes in the CW-EPR hyperfine pattern can be explained by a small decrease in the anisotropy of at least two hyperfine tensors. It is proposed that modifications at the Ca(2+) site may modulate the fine structure tensor of the Mn(III) ion. DFT calculations support the above conclusions. Our data analysis also provides strong support for the notion that in the S(2) state the coordination of the Mn(III) ion is square-pyramidal (5-coordinate) or octahedral (6-coordinate) with tetragonal elongation. In addition, it is shown that only one of the currently published OEC models, the Siegbahn structure [Siegbahn, P. E. M. Acc. Chem. Res.2009, 42, 1871-1880, Pantazis, D. A. et al. Phys. Chem. Chem. Phys.2009, 11, 6788-6798], is consistent with all data presented here. These results provide important information for the structure of the OEC and the water-splitting mechanism. In particular, the 5-coordinate Mn(III) is a potential site for substrate 'water' (H(2)O, OH(-)) binding. Its location within the cuboidal structural unit, as opposed to the external 'dangler' position, may have important consequences for the mechanism of O-O bond formation.     

AMP.PNP affects the dynamical properties of monomer and polymerized actin

Actin is the component of several biological systems and it plays important role in different biological processes, especially in cell motility. The actin-based motility is accompanied with ATP-consume, and the irreversible ATP hydrolysis is coupled with the polymerization of monomer actin into filamentous form. When an actin monomer is incorporated into a filament, the ATPase is activated, and thereby the polymer formation is promoted. The polymer formation and the ATP hydrolysis is associated with internal motions and significant changes of the conformation in reaction partners. In this article, the ATP nucleotide in monomer actin was exchanged by its non-hydrolyzable analogue adenylyl-imidodiphosphate (AMP.PNP), and using two biophysical methods, electron paramagnetic resonance spectroscopy (EPR) and differential scanning calorimetry (DSC), we studied the local and global changes in globular and fibrous actin following the nucleotide exchange. The paramagnetic probe molecule—a maleimide spin label—was attached to Cys-374 site of monomer actin, and its rotational mobility was derived at different temperature. In DSC measurements the transition temperatures of samples with different bound nucleotides were compared. From the measurements we could conclude, that the nucleotide exchange induces changes in the internal rigidity of the actin systems, AMP.PNP-actins showed longer rotational correlation time and increased thermal transition temperature.     

Physical and enzymatic properties of nucleotide-depleted beef heart mitochondrial adenosine triphosphatase.

Tightly bound adenine nucleotides are removed from multiple binding sites on beef heart mitochondrial ATPase (F1) by chromatography on columns of Sephadex equilibrated with 50% glycerol. Release of nucleotides from the enzyme is associated with large decreases in sedimentation velocity (from 11.9 S to 8.4 S) which may be observed in concentrated solutions of polyols. Polyol-induced conformational changes are reversed when the enzyme is returned to dilute buffers. The nucleotide-depleted enzyme restores oxidative phosphorylation in F1-deficient submitochondrial particles. Reconstitution of nucleotide-depleted F1 with the ATP analog (adenylyl-imidodiphosphate (AMP-PNP), almost 5 moles of AMP-PNP per mole of enzyme, results in preparations with substantially inhibited ATPase activity which nevertheless restores oxidative phosphorylation and the 32Pi-ATP exchange reaction in F1-deficient submitochondrial particles. Incubation of the analog-labeled enzyme with ATP and Mg++ results in partial displacement of the analog and a time-dependent recovery of ATPase activity.     

Differential effects of Mg(ii) and N(alpha)-4-tosyl-l-arginine methyl ester hydrochloride on the recognition and catalysis in ATP hydrolysis

The supramolecular interactions of Mg(ii) and N(alpha)-4-tosyl-l-arginine methyl ester hydrochloride (TAME) with ATP have been investigated using (1)H and (31)P NMR spectra. Furthermore, the hydrolysis of ATP catalyzed by Mg(ii) and TAME has been studied at 60 degrees C and pH 7 using (31)P NMR spectra. In the Mg(ii)-ATP-TAME ternary system, the binding interaction of Mg(2+) with ATP involves not only N1 and N7 in the adenine ring but also beta- and gamma-phosphate of ATP. The binding forces are mainly electrostatic interaction and cation (Mg(2+))-pi interaction. The guanidinium group and the aromatic ring of TAME interacts with ATP by beta and gamma phosphate and the adenine ring of ATP. The binding forces are mainly electrostatic interactions and pi-pi stacking. A significant difference between the binary and the ternary system indicates that TAME is essential to the stablization of the intermediate. Kinetic studies show that the hydrolysis rate constant of ATP is 2.16 x 10(-2) h(-1) at pH 7 in the Mg(ii)-TAME-ATP ternary system. The Mg(ii) ion and TAME can accelerate the ATP hydrolysis process. A possible mechanism has been proposed that the hydrolysis occurs through an addition-elimination, in which the phosphoramidate intermediate was observed at 3.21 ppm in the (31)P NMR of the ternary system. These results provide further information concerning the effect of the key amino acid residue and metal ions as cofactors of ATPase on ATP synthesis/hydrolysis at the molecular level.     

The role of the Mg2+ cation in ATPsynthase studied by electron paramagnetic resonance using VO2+ and Mn2+ paramagnetic probes

The electron paramagnetic resonance (EPR), electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectra of Mg2+-depleted chloroplast F1-ATPase substituted with stoichiometric VO2+ are reported. The ESEEM and HYSCORE spectra of the complex are dominated by the hyperfine and quadrupole interactions between the VO2+ paramagnet and two different nitrogen ligands with isotropic hyperfine couplings /A1/ = 4.11 MHz and /A2/ = 6.46 MHz and nuclear quadrupole couplings e2qQ1 approximately 3.89-4.49 MHz and e2qQ2 approximately 1.91-2.20 MHz, respectively. Aminoacid functional groups compatible with these magnetic couplings include a histidine imidazole, the epsilon-NH2 of a lysine residue, and the guanidinium group of an arginine. Consistent with this interpretation, very characteristic correlations are detected in the HYSCORE spectra between the 14N deltaM1 = 2 transitions in the negative quadrant, and also between some of the deltaM1 = 1 transitions in the positive quadrant. The interaction of the substrate and product ADP and ATP nucleotides with the enzyme has been studied in protein complexes where Mg2+ is substituted for Mn2+. Stoichiometric complexes of Mn x ADP and Mn x ATP with the whole enzyme show distinct and specific hyperfine couplings with the 31P atoms of the bonding phosphates in the HYSCORE (ADP, A(31Pbeta) = 5.20 MHz: ATP, A(31Pbeta) = 4.60 MHz and A(31Pgamma) = 5.90 MHz) demonstrating the role of the enzyme active site in positioning the di- or triphosphate chain of the nucleotide for efficient catalysis. When the complexes are formed with the isolated alpha or beta subunits of the enzyme, the HYSCORE spectra are substantially modified, suggesting that in these cases the nucleotide binding site is only partially structured.     

The catalytic Mn2+ sites in the enolase-inhibitor complex: crystallography, single-crystal EPR, and DFT calculations

Crystals of Zn2+/Mn2+ yeast enolase with the inhibitor PhAH (phosphonoacetohydroxamate) were grown under conditions with a slight preference for binding of Zn2+ at the higher affinity site, site I. The structure of the Zn2+/Mn2+-PhAH complex was solved at a resolution of 1.54 A, and the two catalytic metal binding sites, I and II, show only subtle displacement compared to that of the corresponding complex with the native Mg2+ ions. Low-temperature echo-detected high-field (W-band, 95 GHz) EPR (electron paramagnetic resonance) and 1H ENDOR (electron-nuclear double resonance) were carried out on a single crystal, and rotation patterns were acquired in two perpendicular planes. Analysis of the rotation patterns resolved a total of six Mn2+ sites, four symmetry-related sites of one type and two out of the four of the other type. The observation of two chemically inequivalent Mn2+ sites shows that Mn2+ ions populate both sites I and II and the zero-field splitting (ZFS) tensors of the Mn2+ in the two sites were determined. The Mn2+ site with the larger D value was assigned to site I based on the 1H ENDOR spectra, which identified the relevant water ligands. This assignment is consistent with the seemingly larger deviation of site I from octahedral symmetry, compared to that of site II. The ENDOR results gave the coordinates of the protons of two water ligands, and adding them to the crystal structure revealed their involvement in a network of H bonds stabilizing the binding of the metal ions and PhAH. Although specific hyperfine interactions with the inhibitor were not determined, the spectroscopic properties of the Mn2+ in the two sites were consistent with the crystal structure. Density functional theory (DFT) calculations carried out on a cluster representing the catalytic site, with Mn2+ in site I and Zn2+ in site II, and vice versa, gave overestimated D values on the order of the experimental ones, although the larger D value was found for Mn2+ in site II rather than in site I. This discrepancy was attributed to the high sensitivity of the ZFS parameters to the Mn-O bond lengths and orientations, such that small, but significant, differences between the optimized and crystal structures alter the ZFS considerably, well above the difference between the two sites.     

Interaction of bis(1-anilino-8-naphthalenesulfonate) with yeast hexokinase: a steady-state fluorescence study

Bis(1-analino-8-naphthalenesulfonate) (bis-ANS) is a useful probe for hydrophobic areas on protein molecules and it has been proposed that it has a general affinity for the nucleotide binding site(s). There appear to be two different classes of binding sites for bis-ANS on hexokinase and these can be tentatively assigned as primary and secondary binding sites. The rate of binding of bis-ANS at the primary binding site is fast, whereas binding at secondary site(s) is slow. The slow increase in the fluorescence intensity on binding with bis-ANS is not due to conformational change in the enzyme, which may lead to the increase in the quantum yield of the bound dye. Circular dichroism measurements indicate that there is no significant change in the secondary structure on binding with this probe. In the presence of saturating amounts of glucose, the increase in fluorescence intensity due to binding at the secondary binding site(s) is significantly lowered. This indicates that glucose-induced conformational change has been sensed by this probe. From kinetic studies, it has been observed that bis-ANS is an effective competitive inhibitor of yeast hexokinase with respect to ATP. The stoichiometry of binding of this fluorescent probe is about one per subunit at the primary site both in the presence and absence of glucose, and the dissociation constant of bis-ANS is unaffected by glucose. It is possible to decrease significantly the amount of fluorescence intensity at the primary site by nucleotides. These results indicate that bis-ANS interacts at the site where nucleotide interacts. Energy transfer experiments indicate the proximity of some tryptophan(s) and bound bis-ANS molecule(s).     

Conversion of molecular information by luminescent nanointerface self-assembled from amphiphilic Tb(III) complexes

A novel amphiphilic Tb(3+) complex (TbL(+)) having anionic bis(pyridine) arms and a hydrophobic alkyl chain is developed. It spontaneously self-assembles in water and gives stable vesicles that show sensitized luminescence of Tb(3+) ions at neutral pH. This TbL(+) complex is designed to show coordinative unsaturation, i.e., water molecules occupy some of the first coordination spheres and are replaceable upon binding of phosphate ions. These features render TbL(+) self-assembling receptor molecules which show increase in the luminescence intensity upon binding of nucleotides. Upon addition of adenosine triphosphate (ATP), significant amplification of luminescent intensity was observed. On the other hand, ADP showed moderately increased luminescence and almost no enhancement was observed for AMP. Very interestingly, the increase in luminescence intensity observed for ATP and ADP showed sigmoidal dependence on the concentration of added nucleotides. It indicates positive cooperative binding of these nucleotides to TbL(+) complexes preorganized on the vesicle surface. Self-assembly of amphiphilic Tb(3+) receptor complexes provides nanointerfaces which selectively convert and amplify molecular information of high energy phosphates linked by phosphoanhydride bonds into luminescence intensity changes.