Physiochemical characterization of sodium doped zinc oxide nano powder for antimicrobial applications.

Zinc oxide (ZnO) is one of the semiconductor materials with unique antimicrobial properties towards various microorganisms. In this article, pure and Na doped ZnO nanopowders were synthesized by easiest and cost-effective co-precipitation process. X-ray diffraction (XRD),Fourier transform infrared spectroscopy(FT-IR), ultraviolet - visible (UV - Vis) spectroscopy, scanning electron microscopy(SEM), and Energy dispersive X-ray analysis (EDAX) techniques were used to characterize the particle size, surface morphology and chemical composition of prepared materials. The XRD analysis revealed that the samples exhibiting hexagonal wurtzite crystal structure with high crystallinity and the average crystallite size values increased from 23.51 to 28.118 nm. The UV - Vis spectroscopy results exposed that the bandgap energy (Eg) of the samples with the values in the range of 3.068-3.301 eV. The SEM micrographs showed that the morphology of the of synthesized particles are hexagonal and spherical in nanometric size. The EDX spectra confirmed the elemental composition of Na, Zn and O in the crystal lattice and FTIR spectroscopic data proved the formation of functional groups and the presence of chemical bonding at the ZnO interface.Antibacterial activity of pure and Na doped Zinc oxide nanoparticles against Gram-negative pathogenssuch as Escherichia coli, Pseudomonas aeruginosa & Klebsiella pneumoniae and Gram-positive pathogens such as Staphylococcus aureus reveal that the zone of inhibition increases with increasing Na concentration. The antifungal activity against Aspergillus and Candida was investigated.These results demonstrated that the pure and Na doped ZnO samples exhibit enhanced antibacterial and antifungal activity with increasing particle sizein presence of visible light and they could be used as good antibacterial as well as antifungal agents.

A simple and reliable Taguchi approach for multi-objective optimization to identify optimal process parameters in nano-powder-mixed electrical discharge machining of INCONEL800 with copper electrode.

There is a need for heavy-duty machining equipment and tooling to minimize chatter due to work-hardening of the INCONEL materials ahead of cutting. Optimum EDM parameters are to be identified to produce quality products of INCONEL800. Modified Taguchi approach is adopted in the multi-objective optimization to identify the optimum peak current, pulse-on-time and pulse-off-time in the nano powder mixed EDM (n-PMEDM) of INCONEL800 with copper electrode for high material removal rate (MRR) and low surface roughness (SR). Empirical relations for MRR and SR are developed easily in terms of the EDM parameters without use of the MINITAB Release-16 software and validated with test results. Test results are found to be within the expected range. It also demonstrates the advantages of opting Taguchi approach to get complete information through few experiments.

Confirmation of diosmetin 3-O-glucuronide as major metabolite of diosmin in humans, using micro-liquid-chromatography-mass spectrometry and ion mobility mass spectrometry.

Diosmin is a flavonoid often administered in the treatment of chronic venous insufficiency, hemorrhoids, and related affections. Diosmin is rapidly hydrolized in the intestine to its aglicone, diosmetin, which is further metabolized to conjugates. In this study, the development and validations of three new methods for the determination of diosmetin, free and after enzymatic deconjugation, and of its potential glucuronide metabolites, diosmetin-3-O-glucuronide, diosmetin-7-O-glucuronide, and diosmetin-3,7-O-glucuronide from human plasma and urine are presented. First, the quantification of diosmetin, free and after deconjugation, was carried out by high-performance liquid chromatography coupled with tandem mass spectrometry, on an Ascentis RP-Amide column (150 × 2.1 mm, 5 µm), in reversed-phase conditions, after enzymatic digestion. Then glucuronide metabolites from plasma were separated by micro-liquid chromatography coupled with tandem mass spectrometry on a HALO C18 (50 × 0.3 mm, 2.7 µm, 90 Å) column, after solid-phase extraction. Finally, glucuronides from urine were measured using a Discovery HSF5 (100 × 2.1 mm, 5 µm) column, after simple dilution with mobile phase. The methods were validated by assessing linearity, accuracy, precision, low limit of quantification, selectivity, extraction recovery, stability, and matrix effects; results in agreement with regulatory (Food and Drug Administration and European Medicines Agency) guidelines acceptance criteria were obtained in all cases. The methods were applied to a pharmacokinetic study with diosmin (450 mg orally administered tablets). The mean C max of diosmetin in plasma was 6,049.3 ± 5,548.6 pg/mL. A very good correlation between measured diosmetin and glucuronide metabolites concentrations was obtained. Diosmetin-3-O-glucuronide was identified as a major circulating metabolite of diosmetin in plasma and in urine, and this finding was confirmed by supplementary experiments with differential ion-mobility mass spectrometry.

Conformations of nucleotides bound to wild type and Y78F mutant yeast guanylate kinase: proton two-dimensional transferred NOESY measurements.

Wild type and Y78F mutant yeast guanylate kinase (GKy) were studied to investigate the effects of a site-directed mutation on bound substrate conformations. Previously published work showed that Y78 is involved in GMP binding and that the Y78F mutant has 30-fold weaker GMP binding and 2 orders of magnitude less activity, than the wild type. Adenosine conformations of adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphate (ADP) and guanosine conformations of guanosine 5'-monophosphate (GMP) bound to wild type and Y78F mutant yeast guanylate kinase in the complexes GKy x Mg(II)ATP, GKy x Mg(II)ADP, GKy x GMP, and GKy x Mg(II)ADP x [U-13C]GMP were determined by two-dimensional transferred nuclear Overhauser effect (TRNOESY) measurements combined with molecular dynamics simulations. For adenyl nucleotides in wild type complexes, all glycosidic torsion angles, chi, were 54 +/- 5 degrees. In Y78F mutant complexes, adenyl nucleotide glycosidic torsion angles were 55 +/- 5 degrees (GKy x MgATP) and 49 +/- 5 degrees (GKy x MgADP). Thus, the adenyl nucleotides bind similarly for both the wild type and Y78F mutant complexes. However, in the fully constrained, two-substrate complexes, GKy x Mg(II)ADP x [U-13C]GMP, the guanyl glycosidic torsion angle, chi, is 50 +/- 5 degrees with the wild type and 83 +/- 5 degrees with the Y78F mutant. This difference suggests that an unfavorable torsion may be a large part of the mechanism for significantly weaker GMP binding to reaction complexes of the Y78F mutant.

Changes in the 31P NMR spectrum of rabbit muscle myosin subfragment 1. MgADP with temperature.

In pioneering studies on the 31P NMR spectra of MgADP bound to the "molecular motor" myosin subfragment 1 (S1) in the temperature range of 0 to 25 degrees C, Shriver and Sykes [Biochemistry 20 (1981) 2004-2012/6357-6362; Biochemistry 21 (1982) 3022-3028], proposed that MgADP binds to myosin S1 as a mixture of two interconvertible conformers with different chemical shifts for the beta-P resonance of the S1-bound MgADP and that the concentrations of these conformers are related by an equilibrium constant K(T). Their model implied that the weighted average of the chemical shifts of the beta-P(MgADP) for S1-bound MgADP asymptotically approaches a high temperature limit. Here, and in our earlier paper [K. Konno, K. Ue, M. Khoroshev, H., Martinez, B.D. Ray, M.F. Morales, Proc. Natl. Acad. Sci. USA 97 (2000) 1461-1466], we report experimental similarities to Shriver and Sykes, but diverge from them (especially at 0 degrees C) in not finding two distinct peaks and in finding that the average chemical shift does not change with temperature. Our observations can be explained by chemical exchange of beta-P(MgADP) of S1-bound MgADP between two nearly energetically equivalent environments.

Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase. 1. Adenosine conformations by proton two-dimensional transferred nuclear Overhauser effect spectroscopy.

Adenosine conformations of adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate (AMP), and of an ATP analogue, adenylyl imidodiphosphate (AMPPNP), bound to Escherichia coliadenylate kinase (AKe) in the complexes of AKe.Mg(II)ATP, AKe.AMP.Mg(II)GDP, AKe. AMPPNP, and AKe.Mg(II)AMPPNP were determined by transferred two-dimensional nuclear Overhauser effect spectroscopy (TRNOESY) measurements and molecular dynamics simulations. The glycosidic torsion angles, chi, deduced for the adenine nucleotides in these complexes are 51 degrees, 37 degrees, 49 degrees, and 47 degrees, respectively, with an experimental error of about +/-5 degrees. These values are in general agreement with those previously measured for other ATP-utilizing enzymes, suggesting a possible common motif for adenosine recognition and binding. The pseudorotational phase angle, P, of the sugar puckers for the bound nucleotides varied between 50 degrees and 103 degrees. These solution-state conformations are significantly different from those in published data from X-ray crystallography. A computation of the ligand NOEs, made by using the program CORCEMA [Moseley, H. N. B., Curto, E. V., and Krishna, N. R. (1995) J. Magn. Reson. B108, 243-261] with the protein protons in the vicinity of nucleotide included, on the basis of the X-ray structure of the AKe.AMP.AMPPNP complex [Berry, M. B., Meador, B., Bilderback, T., Liang, P., Glaser, M., and Philips, G. N. , Jr. (1994) Proteins: Struct., Funct., Genet. 19, 183-198], showed that polarization transfer to the protein protons does not produce significant errors in the structures determined by considering the ligand NOEs alone.

Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase. 2. 31P and 13C relaxation measurements in the presence of cobalt(II) and manganese(II).

13C spin-lattice relaxation rates have been measured for two complexes of Escherichia coli adenylate kinase (AKe), viz., AKe. [U-(13)C]ATP and AKe.[U-(13)C]AMP.GDP in the presence of the substituent activating paramagnetic cation Mn(II) for the purpose of determination of the enzyme-bound conformations of ATP and AMP. (GDP has been added to the AMP complex with the enzyme in order to hold the cation in the bound complex.) Measurements of relaxation times at three different (13)C frequencies, 181.0, 125.7, and 75.4 MHz, indicate that the relaxation times in the enzyme-nucleotide complexes with the paramagnetic cation are not exchange-limited; i.e. , they are larger than the effective lifetimes of cation binding to these complexes and are, therefore, dependent on the cation-(13)C distances. An analysis of the frequency-dependent relaxation data allowed all of the ten Mn(II)-(13)C distances to be determined in each of the complexes. Similar measurements of the (31)P relaxation rate made on AKe.ATP and AKe.AMP.GDP complexes in the presence of Co(II) as the activating cation yielded Co(II)-(31)P distances for each adenine nucleotide. These distances, together with the interproton distances determined previously from TRNOESY experiments [Lin, Y., and Nageswara Rao, B. D. (2000) Biochemistry 39, 3636-3646], led to a complete characterization of both ATP and AMP conformations in AKe-bound complexes. These conformations differ significantly from the nucleotide conformations in crystals of AKe. AP(5)A and AKe.AMP.AMPPNP as determined by X-ray crystallography.

Paramagnetic effects on nuclear relaxation in enzyme-bound Co(II)-adenine nucleotide complexes: relative contributions of dipolar and scalar interactions.

31P NMR measurements on CoADP bound to creatine kinase designed to estimate the relative contribution of scalar and dipolar interactions to 31P spin relaxation rates show that these rates are primarily due to distance-dependent dipolar interactions and that the contribution of the scalar interaction is negligible.

Mn(II)-EPR measurements of cation binding by aequorin.

Cation binding at 5 degrees C by aequorin, a bioluminescent protein from the jellyfish Aequorea victoria, was examined by means of Mn(II) EPR. The bioluminescence of aequorin is triggered by Ca(II), as well as by trivalent lanthanides, and is inhibited by Mg(II) and Mn(II). Three EF-hand Ca(II)-binding domains have been identified in the aequorin amino acid sequence. In the work reported here, active native aequorin was found to have a single tight binding site for Mn(II) with an association constant of 0.566 microM-1. Ca(II) and La(III) competed for the Mn(II) site with association constants of 1.92 microM-1 and 1.38 microM-1, respectively. The affinity of Ca(II) and La(III) for their two other (presumed) sites on aequorin was an order of magnitude less than their affinity for the Mn(II) site. Mg(II) competed for the Mn(II) site as well but with a much smaller association constant of 0.0109 microM-1. Ca(II)-independent discharged aequorin did not bind Mn(II) to a significant degree. Conjectures on the location of the Mn(II) site in the aequorin amino acid sequence and on the relationship between the binding parameters of the cations and their influence on aequorin activity are given.

31P and 1H NMR studies of the structure of enzyme-bound substrate complexes of lobster muscle arginine kinase: relaxation measurements with Mn(II) and Co(II).

The paramagnetic effects of Mn(II) and Co(II) on the spin-lattice relaxation rates of 31P nuclei of ATP and ADP and of Mn(II) on the spin-lattice relaxation rate of the delta protons of arginine bound to arginine kinase from lobster tail muscle have been measured. Temperature variation of 31P relaxation rates in E.MnADP and E.MnATP yields activation energies (delta E) in the range 6-10 kcal/mol. Thus, the 31P relaxation rates in these complexes are exchange limited and cannot provide structural information. However, the relaxation rates in E.CoADP and E.CoATP exhibit frequency dependence and delta E values in the range 1-2 kcal/mol; i.e., these rates depend upon 31P-Co(II) distances. These distances were calculated to be in the range 3.2-4.5 A, appropriate for direct coordination between Co(II) and the phosphoryl groups. The paramagnetic effect of Mn(II) on the 1H spin-lattice relaxation rate of the delta protons of arginine in the E.MnADP.Arg complex was also measured at three frequencies (viz., 200, 300, and 470 MHz). These 1H experiments were performed in the presence of sufficient excess of arginine to be observable over the protein background but with MnADP exclusively in the enzyme-bound form so that the enhancement in the relaxation rates of the delta protons of arginine arises entirely from the enzyme-bound complex. Both the observed frequency dependence of these rates and the delta E less than or equal to 1.0 +/- 0.3 kcal/mol indicate that this rate depends on the 1H-Mn(II) distances.(ABSTRACT TRUNCATED AT 250 WORDS)

Photochemically-induced dynamic nuclear polarization proton-NMR of aequorin discharged by calcium-independent light emission.

Photochemically-induced dynamic nuclear polarization was used to identify exposed amino-acid residues and to assign resonances in the 1H-NMR spectrum of Ca(II)-independent discharged (inactivated) aequorin. A previous nuclear magnetic resonance, circular dichroism and fluorescence study [Ray, B.D., Ho, S., Kemple, M. D., Prendergast, F. G. and Nageswara Rao, B.D. (1985) Biochemistry 24, 4280-4287] indicated that as the Ca(II)-activated bioluminescent protein aequorin from jellyfish spontaneously emits light in the absence of Ca(II), it changes from a rigid, fully active form to a discharged form in which a number of amino-acid residues are more mobile than in the native protein. Laser-photochemically-induced dynamic nuclear polarization experiments identified tryptophan and tyrosine residues, but not histidine residues, in Ca(II)-independent discharged aequorin to be accessible to the flavin dye used. These exposed residues are also among the mobile residues of the Ca(II)-independent discharged protein. Resonances of all the protons (including the alpha protons) of the accessible tryptophan and tyrosine residues were assigned with the aid of two-dimensional photochemically-induced dynamic nuclear polarization J-correlated spectroscopy. The oxidized chromophore, from which light is emitted in aequorin, was not accessible to the dye in the Ca(II)-independent discharged protein. No exposed residue was detected in the photochemically-induced dynamic nuclear polarization spectrum of Ca(II)-independent discharged aequorin from which the oxidized chromophore was removed, corroborating the previous finding that in this apo-discharged form the protein partially refolds and thereby loses some of the mobility acquired in the formation of the Ca(II)-independent discharged protein.

Structure of metal-nucleotide complexes bound to creatine kinase: 31P NMR measurements using Mn(II) and Co(II).

The structures of metal-nucleotide complexes bound to rabbit muscle creatine kinase have been studied by making measurements of paramagnetic effects of two dissimilar activating paramagnetic cations, Mn(II) and Co(II), on the spin-relaxation rates of the 31P nuclei of ATP and ADP in these complexes. The experiments were performed on enzyme-bound complexes, thereby limiting the contributions to the observed relaxation rate to two exchanging complexes (with and without the cation). Measurements were made as a function of temperature in the range 5-35 degrees C and at three 31P NMR frequencies, 81, 121.5, and 190.2 MHz, in order to determine the effect of exchange on the observed relaxation rates. The relaxation rates in E X MnADP and E X MnATP are independent of frequency, and their temperature variation yields activation energies (delta E) in the range 5-8 kcal/mol; in the transition-state analogue complex E X MnADP X NO3- X Cre (Cre is creatine), delta E is increased to 17.3 kcal/mol. These results demonstrate that the relaxation rates in the Mn(II) complexes are exchange limited and are incapable of providing structural data. It is shown further that use of line-width measurements to estimate the lifetime of the paramagnetic complex leads to incorrect results. The relaxation rates in E X CoADP and E X CoATP exhibit frequency dependence and delta E values in the range 1-3 kcal/mol; i.e., these rates depend on the Co(II)-31P distances, whereas those in the E X CoADP X NO3- X Cre complex have delta E approximately 18 kcal/mol and are significantly contributed by exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Proton NMR of aequorin. Structural changes concomitant with calcium-independent light emission.

Aequorin, a Ca(II)-sensitive bioluminescent protein from jellyfish, emits light at 469 nm from an excited state of a substituted pyrazine (oxyluciferin) which results from the oxidation of a chromophore molecule that is noncovalently bound to the protein. The chromophore is oxidized when Ca(II) or other activating metal ions are bound by aequorin. In the absence of Ca(II), spontaneous emission of light, referred to as Ca(II)-independent light emission, occurs at a rate less than 10(-6) of that for Ca(II)-induced emission. Proton nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence were used to study structural changes of aequorin accompanying Ca(II)-independent light emission. Time course studies by 1H NMR and CD demonstrate that as a result of Ca(II)-independent light emission, aequorin progressively changes from a rigid, fully active form showing little segmental mobility to a practically unfolded, discharged (i.e., inactive) form in which a number of amino acid residues are significantly mobile. This slow discharged protein (SDP) is distinct in nature and conformation from aequorin which has been discharged by Ca(II), i.e., the blue fluorescent protein. The rate of Ca(II)-independent discharge of aequorin is substantially reduced in the presence of excess Mg(II); the time constant for inactivation at 5 degrees C is 30 days with no Mg(II) present and 70 days with Mg(II) present. The NMR spectra are nearly identical at a given stage of inactivation whether or not Mg(II) is present. Oxyluciferin remains bound to SDP. If it is removed, however, by column chromatography, the resulting apo-SDP partially refolds, and the segmental mobility acquired in the formation of SDP is significantly attenuated particularly for some of the aromatic amino acid residues.

31P NMR lineshapes of beta-P (ATP) in the presence of Mg2+ and Ca2+: estimate of exchange rates.

The 31P NMR chemical shift of beta-P of adenosine triphosphate (ATP) undergoes a substantial change (approximately 2-3 ppm) upon chelation of divalent ions such as Mg2+ or Ca2+. In the presence of nonsaturating amounts of Mg2+ or Ca2+, the lineshape of this resonance depends on the characteristic association and dissociation rates of these metal-ATP complexes. A procedure for computer simulation of this lineshape is outlined. A comparison of computer-simulated lineshapes with the experimental lineshapes obtained at 121 MHz was used to determine the following dissociation rate of Mg2+ and Ca2+ from their ATP complexes at 20 degrees C and pH 8.0: Ca2+, greater than 3 X 10(5) s-1 (Hepes buffer); Mg2+, 1200 s-1 (no buffer), 1000 s-1 (Tris buffer) and 2100 s-1 (Hepes buffer). The limits of error are +/- 10% in these values. For the Mg2+ complexes, the rates were determined as a function of temperature to obtain activation energies (with a maximum deviation of 10% in the least-squares fit): 8.1 Kcal/mole (no buffer and Hepes buffer) and 6.8 kcal/mole (Tris buffer). Lineshapes of the beta-P resonance simulated as a function of Mg2+ concentration, using 2100 s-1 for the dissociation rate, are also presented. The computer simulation of lineshapes offers a reliable and straightforward method for the determination of exchange rates of diamagnetic cations from their ATP complexes, under a variety of sample conditions.

Analysis of 31P NMR spectra of enzyme-bound reactants and products of adenylate kinase using density matrix theory of chemical exchange.

31P NMR spectra of equilibrium mixtures of enzyme-bound reactants and products of the adenylate kinase reaction (formula; see text) were analyzed by using computer simulations based on density matrix theory of chemical exchange. Since adenylate kinase has the unique feature that the reactants in the reverse direction are both ADP molecules, which are indistinguishable off the enzyme, the density matrix equations are formulated for the ABC + D in equilibrium A'B' + A"B" exchange appropriate for the reaction, in which the interchange of A'B' and A"B" is explicitly introduced. It is shown that the consideration of this interchange is essential to explain the experimentally observed line shapes. By comparison of the computer-simulated spectra with various values for the rates of the exchange with the experimental spectra for porcine adenylate kinase at pH 7.0 and T = 4 degrees C, the following characteristic rates were determined: interconversion rates, 375 +/- 30 s-1 (ATP formation) and 600 +/- 50 s-1 (ADP formation); interchange rates of donor and acceptor ADP's, 100 +/- 30 s-1 (in the presence of optimal Mg2+ concentration), 1500 +/- 100 s-1 (in the absence of Mg2+). It is shown that under the conditions of the experiments the interchange rate is the lower limit of the dissociation rate of ADP (or MgADP from the acceptor site if Mg2+ was present) from the enzyme complexes. The significance of these interchange rates and their values relative to the interconversion rates is discussed with special reference to the role of the Mg2+ ion in the differentiation of the two nucleotide binding sites on adenylate kinase.

31P NMR of enzyme-bound substrates of rabbit muscle creatine kinase. Equilibrium constants, interconversion rates, and NMR parameters of enzyme-bound complexes.

The reaction catalyzed by rabbit muscle creatine kinase ATP + creatine in equilibrium ADP + P-creatine has been investigated by 31P NMR. At pH 8.0 and 4 degrees C, the equilibrium constant of the overall reaction [P1][P2]/[S1] [S2] is found to be 0.08, while that for the interconversion step between enzyme-bound substrates and products [E.P1. P2]/[E.S1.S2] is estimated to be approximately 1; the latter value is the same for all other kinases investigated. The rate of interconversion of enzyme-bound substrates and products is approximately 90 s-1 and is not the rate-limiting step of the overall reaction. Of the phosphate groups in enzyme complexes of reactants or products, the 31P chemical shifts of beta-P(ADP) and beta-P[MgADP) change by approximately 2 ppm downfield while all others change by less than 0.8 ppm. In the transition state analog complexes E.MgADP.NO3-.creatine and E.MgADP.HCOO-.creatine, the beta-P(MgADP) signal shows a substantial upfield shift in the direction of the beta-P(MgATP) resonance. The pattern of chemical shifts and line shapes of nucleotide complexes of creatine kinase parallel those for the corresponding complexes of arginine kinase, indicating structural and/or conformational similarity of the phosphate chains of nucleotides bound to the two enzymes. However, a difference in active sites is indicated by the pH independence (pH 6.0 to 9.0) of the chemical shift of the beta-P of MgADP bound to creatine kinase, whereas with arginine kinase this resonance showed a pKa approximately 7.5.

Asymmetric binding of the inhibitor di(adenosine-5') pentaphosphate (Ap5A) to adenylate kinase.

The effect of binding diadenosine pentaphosphate (Ap(5)A) to adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3) has been investigated by (31)P nuclear magnetic resonance. The symmetric molecule, Ap(5)A, is a potent inhibitor of the adenylate kinase reaction, 2 ADP right arrow over left arrow ATP + AMP. Free Ap(5)A has two groups of signals in its (31)P nuclear magnetic resonance spectrum centered at 11.1 and 22.8 parts/million (ppm) upfield from 85% H(3)PO(4) that are assigned to the end (1-P and 5-P) and middle (2-, 3-, and 4-P) phosphates, respectively. Addition of Mg(2+) shifts the centers of these resonances to 11.7 and 22.3 ppm. The spectrum of Ap(5)A bound to porcine adenylate kinase shows five groups of signals centered at 10.9, 11.9, 20.5, 22.7, and 24.0 ppm; the resonances at 11.1 ppm (1-P and 5-P) and at 22.8 ppm (2-P and 4-P) are now clearly split, indicating asymmetric binding of Ap(5)A to the enzyme. The asymmetry is strikingly enhanced in enzyme-bound MgAp(5)A, which has resonances at 10.5, 12.5, 18.6, 22.7, and 25.6 ppm. By the addition of Mn(2+) to the enzyme.MgAp(5)A complex, the observed signals in increasing order of shifts were tentatively assigned to 1-P, 5-P, 4-P, 3-P, and 2-P, where the 3-, 4-, and 5-P positions correspond to the ATP-binding site on the enzyme. The asymmetry introduced in the phosphate chain of enzyme.MgAp(5)A is indicated by the (31)P chemical shift of 7 ppm between 2- and 4-P, which is one of the largest thus far observed for phosphate substrates bound noncovalently to enzymes.