Water Proton Relaxation Rate Research Articles

Tissue distribution and stability of metalloporphyrin MRI contrast agents.

Mn(III), Fe(III), and Gd(III) complexes of tetrakis(4-sulfonatophenyl)porphyrin (TPPS) and several other porphyrins were evaluated as potential MRI contrast agents. Based on consideration of relaxivity and stability properties in solution, MnTPPS was found to be the compound of choice. At pH 7 the Gd and Mn complexes significantly enhanced the water proton relaxation rate, while the relaxivity of FeTPPS exhibited a significant loss of relaxivity above pH 6 due to oxy-dimer formation. Although GdTPPS exhibited the highest relaxivity in solution, this property was rapidly lost due to dissociation of the metal ion. By contrast MnTPPS remained stable in human plasma after incubation for 9 days. Upon intravenous injection into athymic mice bearing subcutaneous human colon carcinoma xenografts, MnTPPS provided enhanced relaxation of the tissue water in several excised mouse tissues, notably kidney, liver, and tumor. The results at a fixed field (0.25 T) and relaxation dispersion studies showed decreases in water relaxation rates with time for kidney and liver, but an increase for the tumor, with a maximum near 4 days at the highest dose used.

Molecular dynamics in the system water-dimethylsulphoxide

N.M.R. proton relaxation rates of normal and 17O-enriched water and deuteron relaxation rates of heavy water were measured in mixtures with dimethylsulphoxide at different compositions and temperatures. The 17O-induced proton relaxation rate was extracted and from this the rotational correlation time of water was determined. Using these correlation times the composition-dependence of the deuterium quadrupole coupling constant of water was derived and a strong variation over the measured composition range is evident.

A proton NMR relaxation evaluation of a model of brain oedema fluid

Using CSF/ protein solutions as models of vasogenic extracellular fluid, the concentration dependences of longitudinal and transverse proton relaxation rates have been measured at 37 degrees C and 100 MHz for the serum proteins, albumin, IgG, a 2.8:1 mixture of albumin and IgG and human serum itself. These measurements have been used to determine the sensitivity of proton relaxation rates to the amount and the type of the protein in oedema fluid, and to establish the significance of both of these factors in relation to a separation of vasogenic from cytotoxic oedema. An extension of the two-environment rapid-exchange model of solvent relaxation is presented, which account for the measurements on protein mixtures and which enables estimates to be made of the number and the transverse relaxation rate of water protons associated with the protein surface.

Metal ion binding to tetrameric lima bean lectin.

The binding of Mn2+ and Ca2+ to tetrameric lima bean lectin has been examined by equilibrium dialysis and magnetic resonance techniques. Demetalized lectin prepared by acid treatment binds either 1 Mn2+ or 2 Ca2+/monomer. When demetalized lectin is presaturated with Ca2+, only 1 Mn2+ binds per dimer. Water proton relaxation rate enhancements and Mn2+ electron spin resonance spectra were used to monitor metal ion association processes. Following Mn2+ binding to demetalized lectin, a conformational change with activation energy of 16 kcal/mol was detected; this is similar in magnitude to that observed for a conformational change with the lectin concanavalin A. The pH dependence suggests that a histidine residue is involved. ESR spectroscopy shows clearly that 1 Mn2+ binds to each demetalized subunit, but that Ca2+ induces dissociation of half the Mn2+; this result is in agreement with the equilibrium dialysis studies.

Proton Longitudinal Relaxation Rates of Water in Aqueous Orthophosphoric Acid

Abstract Longitudinal relaxation rates of water protons were determined for aqueous solutions of orthophosphoric acid at concentrations up to 85% by weight, and their concentration dependence was investigated. An appropriate choice of variables yielded an essentially linear plot for concentrations up to at least 15% by weight. Deviations from linearity are discussed and are interpreted in terms of mass-law effects. The use of parameters of this plot for deriving values for correlation coefficients and for a hydration parameter is illustrated by application to observed data and data from the literature.

Metal ion binding properties of hen ovalbumin and S-ovalbumin: characterization of the metal ion binding site by 31P NMR and water proton relaxation rate enhancements.

In this study, water proton relaxation rate (PRR) enhancements have been used to characterize the binding of metal ions to native ovalbumin, ovalbumin in which phosphate has been enzymatically cleaved from one or both of the two protein phosphoserines, and a heat-stabilized form of the protein (S-ovalbumin). With Scatchard plots constructed from water PRR enhancements, it was found that native ovalbumin and S-ovalbumin had one strong binding site for Mn2+ ion (KD approximately equal to 6.0 X 10(-4) M). Alkaline phosphatase treated ovalbumin, a protein having a single phosphoserine, had one Mn2+ binding site of slightly weaker affinity (KD approximately equal to 8.3 X 10(-4) M), while acid phosphatase treated ovalbumin, a dephosphorylated protein, had two much weaker Mn2+ ion binding sites (KD approximately equal to 1.3 X 10(-3) M). Competitive binding studies on the native protein suggested that Zn2+ ion competes with Mn2+ for the single strong-affinity site (KD approximately equal to 6.1 X 10(-3) M) while Mg2+ and Ca2+ do not. In a second set of experiments, the paramagnetic contribution to the 31P spin-lattice (T1P) and spin-spin (T2P) relaxation times at three separate magnetic field strengths was measured. Correlation times tau c characterizing Mn2+-31P dipolar relaxation were estimated from the ratios of T1P/T2P at a single field and from the ratios of spin-lattice relaxation rates at three different field strengths. The correlation times so obtained, ranging from about 0.7 to 7.7 ns at the three field strengths, were used in calculating distances from the bound Mn2+ ion to the phosphoserines of native ovalbumin, S-ovalbumin, and alkaline phosphatase treated ovalbumins. It was determined that the phosphate of phosphoserine-68 was 5.95 +/- 0.26 and 6.29 +/- 0.18 A from the Mn2+ in the native and alkaline phosphatase treated protein, respectively, and 6.99 +/- 0.30 A away from the Mn2+ in S-ovalbumin. The phosphate of phosphoserine-344 was determined to be 5.31 +/- 0.20 and 5.75 +/- 0.10 A from the Mn2+ ion in native ovalbumin and S-ovalbumin, respectively. The 13C nucleus of [1-13C]galactose enzymatically transferred to the nonreducing end of the ovalbumin oligosaccharide chain was not found to be significantly relaxed by Mn2+ bound to the protein, even at 1:1 stoichiometric ratio of metal:protein. Using this, we estimate the nonreducing terminal of the ovalbumin oligosaccharide to be at least 39 A from the metal ion binding site on the protein.

Proton relaxation rates of water in dilute solutions of β-lactoglobulin. Determination of cross relaxation and correlation with structural changes by the use of two genetic variants of a self-associating globular protein

In order to relate resonance relaxation behavior to protein structural states, pulse Fourier transform NMR was employed to obtain water proton longitudinal and transverse relaxation rate ( R 1 and R 1 ϱ = R 2) of bovine β-lactoglobulins A and B in buffered solutions. Measurements at concentrations from 5 to 100 mg/ml were made at pH 4.65, 6.2 and 8.0, at 30 and 2°C, to monitor specific structural changes. The parameters characterizing the concentration dependence of the observed R 1 and R 2 were used to derive a number of quantities relating to protein-influenced water, including a hydration parameter h. Changes in h under the different sets of conditions were correlated with (a) the irreversible denaturation of this protein at pH 8.0, 2°C and (b) the dimer ⇄ octamer association at pH 4.65, 2°C. Corresponding correlation times, however, were low, indicating cross relaxation which had not manifested itself as nonexponential relaxation because of the large amount of water present. Differences in the extent of the ⇄ octamer association between genetic variants A and B allowed an evaluation of dynamics and extent of hydration from R 2 alone, assuming the absence of intermolecular interactions. Derived parameters were in agreement with hydrodynamic and X-ray values in the literature. Cross relaxation was likewise evaluated and was found to contribute to R 1 to a large extent. The results show that changes in proton relaxation rates in solutions of a globular protein occuring as genetic variants with different physical properties (such as β-lactoglobulin) can be utilized to detect variations in hydration corresponding to changes in molecular association and conformation, as well as to obtain cross relaxation and structural data.

Determinants of proton relaxation rates in tissue.

It is well established that the longitudinal magnetic relaxation rate of solvent water protons, 1/T1, increases markedly in homogeneous protein solutions as the magnetic field is reduced well below the traditional NMR range. For a 5% solution of protein of 10(5) Da, for example, 1/T1 increases from about 50% above the pure water rate of 20 MHz to five times the water rate at 0.01 MHz. For tissue, including blood, the behavior is similar. Data for blood show that extracellular water has ready access to the hemoglobin inside red blood cells, which causes the enhanced relaxation. The extent to which cell water can sample the spatial structure of soft tissue, and how this structure influences relaxation rates, is as yet unknown. Nonetheless, the relaxation data for tissue can be accommodated within the conceptual framework developed earlier for analyzing homogeneous solutions of diamagnetic proteins. The variation of 1/T1 with field differs among tissues, and its magnitude at a given field can vary by more than a factor of three, far more than does the water content of the tissues. Solute complexes of paramagnetic ions with macromolecules, which increase the relaxation rates of solvent protons, can be introduced intravenously in tissue. They are known to accumulate in specific organs, and therefore have potential utility as contrast-enhancing agents in NMR imaging. Mn2+ and Gd3+, for example, produce characteristic dependences of 1/T1 on magnetic field that vary with the chemical state of the agent. The possibility exists, therefore, for monitoring the in vivo biochemistry of these agents.

Kinetic and spectroscopic studies of the interaction of copper with dopamine beta-hydroxylase.

The question of the stoichiometry of copper bound to dopamine beta-hydroxylase and the number of copper atoms required for maximal activity was addressed in this study. Incubation of tetrameric enzyme from bovine adrenal medulla with 64Cu2+ followed by rapid gel filtration yielded an enzyme containing 8.3-8.9 mol of Cu/mol of tetramer. An identical stoichiometry was obtained by analysis of bound copper by atomic absorption methods. NMR and EPR were used to monitor titrations of the enzyme with Cu2+ and showed that the longitudinal relaxation rate of solvent water protons and the amplitude of the signal at g approximately 2 increased linearly up to a copper to protein ratio of approximately 8. Additional titrations also indicate that an enzyme-Cu2+-tyramine-CN- inhibitory complex was formed when 8 mol of Cu2+ are bound per mol of enzyme. The rate of inactivation of dopamine beta-hydroxylase by the mechanism-based inhibitor 2-Br-3-(p-hydroxyphenyl)-1-propene was measured and used as a method to follow enzymatic catalysis. An increase in rate was observed with increasing Cu2+ up to a protein to Cu2+ ratio of 8 Cu/tetramer. The rate becomes constant after this ratio is achieved. These data indicate that dopamine beta-hydroxylase specifically binds 8 mol of Cu/tetramer and that this stoichiometry is required for maximal activity.

Effects of physical and chemical treatments on chloroplast manganese. NMR and ESR studies

Measurements were made of the water proton relaxation rate ( T −1 2 = R 2), electron spin resonance (ESR) six-line signal of ‘free’ Mn 2+, and O 2-evolution activity in thylakoid membranes from pea leaves. The main results are: (1) Aging of thylakoids at 35°C causes a parallel decrease in O 2-evolution activity, in R 2 and in the content of bound Mn, suggesting that R 2 may be related to the loosely bound Mn involved in O 2 evolution. (2) Treatment of thylakoids with tetraphenylboron (TPB) at [TPB] > 2 mM produces a 2-fold increase in R 2, without release of Mn 2+. The titration curve exhibits three sharp end points. The first end point occurs at a [ TPB] [ chlorophyll] of 1.25, at which the O 2 evolution is completely inhibited. (3) Treatment of thylakoids with NH 2OH also increases R 2 by nearly 2-fold, either by the reduction of the higher oxidation states of Mn to Mn 2+ and / or by exposing the Mn to solvent protons. Also, progressive release of bound Mn occurs at [NH 2OH] ≥ 1 mM as shown by an increase increase in the Mn 2+ ESR signal and a decrease in R 2. (4) Addition of H 2O 2 (0.1–1.0%) to thylakoids causes an enhancement of R 2 similar to that by NH 2OH, but without the release of Mn 2+. (5) Heat treatment of thylakoids at 40–50°C releases Mn 2+ and increases R 2. Conversely, pH values of 7 to 4 release Mn 2+ without changing R 2 while pH values of 7–9 increase R 2 without releasing Mn 2+. Thus, both high and low pH values as well as the heat treatment cause structural changes enhancing the relaxivity of the bound Mn or of other paramagnetic species.

Investigation of the system copper(II) carbonic anhydrase and HCO 3−1/CO 2

Copper(II) substituted human and bovine carbonic anhydrases B in the presence of bicarbonate have been investigated in solution through water-solvent proton nuclear magnetic resonance (nmr) at variable magnetic fields. HCO 3 -, contrary to all the other monoanionic inhibitors, partially reduces the water proton relaxation rates. This has been accounted for on the basis of the availability within the active cavity of two coordination positions partially overlapping 13C-nmr measurements on both CO 2 and HCO 3 - confirm that HCO 3 - binds the metal, whereas CO 2 interacts with the paramagnetic center at nonbonding distance. The upper limit for the CO 2 α HCO 3 - interconversion has been estimated to be 10 sec −1.

Environment of Mn(III) and its function in a red-violet colored acid phosphatase

Purple colored metal-containing acid phosphatases have been purified from beef spleen, uterine fluid, sweet potato, soybean, and spinach leaves [1–3]. These acid phosphatases are divided into subgroups of Mn-containing acid phosphatases from some plants and Fe-containing acid phosphatases from mammalian organs. We have isolated and crystallized Kintoki sweet potato tubers [4] Atomic absorption analyses indicated the presence of one Mn ion per one mol of enzyme molecule (MW 110,000). This red-violet Mn-containing acid phosphatase gave an intense absorption maximum at 515 nm (ϵ = 2460) and a CD extreme at 555 nm (Δϵ = −0.53), respectively. The ratio of Δϵ/ϵ (Kuhn's anisotropic factor) was 2 × 10 −4 for the characteristic visible band. The value is typical of an electrically allowed charge-transfer transition. No ESR signals were detected in the native enzyme at 293 and 77 K. In contrast, the acid- and heat-treated colorless enzyme showed characteristics six-line ESR patterns, around g = 2, based on the aquated Mn(II) ion (I = 5/2). The results is reasonable if it is assumed that the violet chromophore represents as ESR-silent form of the metal, Mn(III). In addition this enzyme showed the absorption band at 1160 nm, which can be assigned to a transition 5A– 5B of high-spin Mn(III). The intense 515 nm band was also assigned to an electrically allowed charge-transfer band from the ligand to the metal, which was expected in Mn(III)(d 4) rather than Mn(II)(d 5). The resonance Raman spectrum of the native enzyme, excited by the 5145 Å line of argon ion laser, exhibited prominent Raman lines at 1230, 1289, 1508, and 1620 cm −1. Similar resonance Raman lines have been reported in Mn(III)-ovotransferrin (1173, 1264, 1501, and 1603 cm −1), Fe(III)-transferrin (1174, 1264, 1508, and 1613 cm −1) and uteroferrin (1177, 1265, 1505, and 1607 cm −1). In these Mn(III)- and Fe(III)-proteins to the four characteristic Raman lines have been assigned to the vibration of the coordinated phenolate anion. Accordingly, the resonance Raman spectrum of the Mn(III)-containing acid phosphatase is interpreted in terms of the internal vibrations of a coordinated phenolate anion [5]. In the shorter wave number region (<1000 cm −1), the resonance Raman spectra of the native enzyme were obscured by fluorescent background, which is due to the tryptophan residues. Tryptophan modification of the enzyme by N-bromosuccinimide showed a marked decrease of fluorescence and the N-bromosuccinimide-treated enzyme exhibited a positive resonance Raman band at 370 cm −1. Such a Mn(III)S stretching mode at approximately 370 cm −1 was reported in the infrared spectrum of the tris(N,N-diethyl dithiocarbamato) Mn(III) complex. Symmetric stretching vibrations of sulfhydryl sulfur to Fe(III) bonds have been assigned at 315–365 cm −1 in iron sulfur proteins and synthetic ironsulfur clusters. Therefore, the resonance Raman line at 370 cm −1 is assigned to Mn(III)z.sbnd;S(cysteine) stretching mode [6]. In the Mn(III)-containing acid phosphatase the enzyme activity was reduced in parallel with a decrease in the 515 nm absorption attributed to the Mn ion directly coordinated with some amino acid residues. Mn(III)-chelating reagents strongly inhibited the phosphatase activity and decreased PRR (proton relaxation rate of water) enhancement of enzyme solution. 31P NMR spectra of the enzyme in the presence of phosphate as substrate showed the pronounced broadening (full width at half-height: 37.8 ppm) 31P phosphate resonance line. The broadening effect corresponded well to 31P NMR signal of the model pyrophosphateMn(III) complex (full width at half-height: 36.2 ppm). Addition of phosphate to this enzyme solution decreased PRR enhancement by 25%. These evidences indicated strongly that Mn(III) is located on the active center in the enzyme, binds to phosphate esters, and plays an essential role in the catalytic reaction of hydrolysis of phosphomonoesters. In conclusion, (1) only single Mn ion per one enzyme molecule is contained in the enzyme, (2) the trivalenT Mn is coordinated by tyrosine and cysteine residues of the protein molecule, and (3) phosphate binds to the Mn(III) active center of this enzyme. Acid phosphatase uses Mn(III) ion to induce effective binding of phosphate substrate in an acidic environment. In our latest experimental results we note similarities and differences between plant's Mn(III)- and mammalian Fe(III)-containing acid phosphatases.

Divalent metal ions in the pharmacodynamics of morphine-like opiates

Paramagnetic Mn(II) ions were used to sample the interaction of morphine-like opiates with divalent metal ions, which are known, especially Ca ++, to be involved in the mechanisms of drug dependence and tolerence. The motional correlation time of the opiate molecule in aqueous solution was determined by selective proton irradiation methods (τ c = 7 × 10 −10 sec at 22 °C) and a preferred cis conformation of H 5 and H 6 protons was inferred. The water proton relaxation rates at different metal to ligand ratios were used to evaluate the equilibrium constant (K ass = 10 −2 M −1) by assuming a dipolar only interaction with T −1 1p modulated by rotational tumbling of the complex (see figure). The temperature dependence of T −1 1p was measured (Table I) in the range 25–60 °C: it turned out that H 1, H 2, H 5, H 7 and H 8 proton relaxation rates were in the fast exchange region, while the −NCH 3 protons were undergoing slow exchange from the metal coordination sphere. ▪ t001 Temperature Dependence of T −1 1p of Morphine Protons. [Morphine] = 0.05 M, [Mn ++] = 0.1 m M, pD = 7. 1000/ T (K −1) T −1 1p (sec −1) H 5 −NCH 3 3.00 0.10 0.24 3.10 0.22 0.13 3.20 0.37 0.05 3.25 0.42 0.04 3.30 0.50 0.07 3.35 0.56 0.12 As consequence, metal-proton distances could be calculated for almost all the protons considered and it was shown that simultaneous binding to the two −OH groups was consistent with the similar measured magnitudes of the MnH 1, MnH 2, MnH 7 and MnH 8 vectors (r is in the range 3.01 – 3.16 Å). H 5 was found to be the nearest proton to the metal ion ( r = 2.84 Å). The temperature dependence of T − 1p of the -NCH 3 methyl protons was taken to evaluate the thermodynamic functions for the exchange process from the metal coordination sphere (k off = 2.25 × 10 4 sec −1, Δ H ≠ = 14.1 kcal/mol, ΔS ≠ = 7.2 e.u.). The structural and kinetic information was used to suggest that interaction with divalent metal ions is characterized, for morphine-like opiates, by an almost exclusively enthalpic barrier due to binding to charged OH groups. Such interaction can be relevant either in modulating the interaction of divalent metal ions with external groups in membranes or in affecting the receptor-opiate interaction modes.

Binding of Mn(II) ions to lecithin bilayers as determined by ESR and NMR investigations

The Mn(II)-lecithin system was investigated by means of paramagnetic relaxation studies. Unsonicated and sonicated aqueous dispersions were considered at various temperatures andpH values. Information was derived from both the frequency dependence of the ESR line shape and the paramagnetic contributions to the water proton relaxation rates. A dynamic equilibrium was suggested, by taking into account the role of the through-water cation binding in the metal-lipid interaction.

Water Relaxation Measurements on Semiquinones of Various Flavoproteins

AbstractThe water relaxation rates of several flavoproteins in the semiquinone state have been investigated by the spin echo technique. The results indicate a rather unspecific interaction between water and the protein‐bound flavosemiquinones. An average interaction distance of 0.3‐0.5 nm has been estimated. From the temperature dependence of the rate constants the free energy of activation for proton exchange is calculated to be about 17 kJ/mol. The rate of proton exchange is around 1011 s−1 for the flavosemiquinones investigated are accessible to water regardless of their ionic state.The large difference in relaxation rates of water protons between D‐ and L‐ amino‐acid oxidases is noticeable. Oxynitrilase exhibits the highest whereas Azotobacter vinelandii flavodoxin shows the lowest water relaxation rate of the flavoproteins studied. The results are discussed in relation to the visible‐light absorption properties of the flavoproteins.

1H-NMR relaxation studies of native and thermally denatured lysozyme solutions : An isotope dilution experiment

1H-NMR relaxation times are reported for native and thermally denatured lysozyme aqueous solutions measured as the function of the proton mole fraction in the sample. A two-exponential character of proton longitudinal relaxation function was observed for native lysozyme solutions: the fast component was attributed to the non-exchangeable protein protons, the slow one to water protons. Purely exponential decay of longitudinal magnetization was observed for the thermally denatured samples. This has been explained in terms of a fast spin exchange model. The contributions of the protein protons to the water proton relaxation rate in native and thermally denatured samples were determined, too.

The interaction of sulfate with carbonic anhydrase.

In the absence of sulfate the pH/rate profile for the 4‐nitrophenyl acetate hydrolase activity of bovine carbonic anhydrase is complex. The results fit with a microscopic ionization scheme involving two electrostatically interacting groups. The activity depends on the concentration of the basic form of one of these groups. Proton NMR spectra show that the active site residue, His‐64, has titration behaviour corresponding to that of the second group of the ionization scheme. The addition of increasing concentrations of Na2SO4 gradually converts the pH/rate profile to that of a simple titration curve. A pKa of 6.9 is found at 50 mM Na2SO4. Concomitantly the titration curve of His‐64 changes. The results fit with the microscopic ionization scheme if it is assumed that significant SO2−4binding occurs only when both His‐64 and the activity‐linked group are protonated. At constant pH, sulfate behaves as if it inhibits the enzyme only partially. However, data are presented suggesting that the enzyme‐sulfate complex is inactive, but the binding of SO2−4depends strongly on ionic strength. Thus, above about 25 mM sulfate any further increase of the sulfate concentration is nearly compensated by a decrease of the binding affinity. The binding of a monovalent anion, C1−, also depends on ionic strength, but this dependence is much less prominent than for SO2‐4. Sulfate shows non‐competitive behaviour with respect to CO2 and appears to compete with HCO−3. Iodide and sulfate appear to be mutually competitive. The low pH limit of Ki for I−is 3 mM and about 0.1 mM in the presence of 50 mM Na2SO4 and in the absence of sulfate respectively. In the absence of sulfate the Co(II)‐substituted bovine enzyme shows a complex pH/rate profile similar to that of the native enzyme. Changes of the optical spectrum parallel those of the activity. Relaxation rates (T−11)of water protons in the presence of Co(II) enzyme indicate that the Co(II)ion might have a H2O ligand at low pH in sulfate‐free solutions. The effects on sulfate of human carbonic anhydrase II (or C) are similar to those on the bovine enzyme. The effects of sulfate on the human 1 (or B) enzyme are smaller.

Oxygenation dependence of the transverse relaxation time of water protons in whole blood at high field

At high and medium magnetic field, the transverse NMR relaxation rate ( T 2 −1) of water protons in blood is determined predominantly by the oxygenation state of haemoglobin. T 2 −1 dependes quadratically on the field strength and on the proportion of haemoglobin that is deoxygenated. Deoxygenation increases the volume magnetic susceptibility within the erythrocytes and thus creates local field gradients around these cells. From volume susceptibility measurements and the dependence of T 2 −1 on the pulse rate in the Carr-Purcell-Meiboom-Gill experiment, we show that the increase in T 2 −1 with increasing blood deoxygenation arises from diffusion of water through these field gradients.

Divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase.

Four different techniques, equilibrium dialysis, protection of enzymatic activity against chemical inactivation, 31P relaxation rats, and water proton relaxation rates, are used to study divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase, EC 3.6.1.1. A major new finding is that the binding of a third divalent metal ion per subunit, which has elsewhere been implicated as being necessary for enzymatic activity [Springs, B., Welsh, K. M., & Cooperman, B. S. (1981) Biochemistry (in press)], only becomes evident in the presence of added inorganic phosphate and that, reciprocally, inorganic phosphate binding to both its high- and low-affinity sites on the enzyme is markedly enhanced in the presence of divalent metal ions, with Mn2+ causing an especially large increase in affinity. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide evidence against divalent metal ion inner sphere binding to phosphate for enzyme subunits having one or two divalent metal ions bound per subunit and evidence for a conformational change restricting active-site accessibility to solvent on the binding of a third divalent metal ion per subunit.

Magnetic resonance studies on interaction of bovine brain hexokinase with manganous ion, glucose, and glucose 6-phosphate

Bovine brain hexokinase enhances the effect of Mn(II) on the longitudinal relaxation rate of water protons. Direct interaction of Mn(II) with the enzyme has been studied using electron spin resonance and proton relaxation rate enhancement methods. The results indicate that brain hexokinase has 1.05 ± 0.13 tight binding sites and 7 ± 2 weak binding sites with a dissociation constant, K D = 25 ± 4 μM and K′ D = 1050 ± 290 μM, respectively, at pH 8.0, 23 °C. The characteristic enhancement ϵ b) for hexokinase-Mn(II) complex evaluated from proton relaxation rate enhancement studies, gave ϵ b = 3.5 ± 0.4 for tight binding sites and an average ϵ b = 2.3 ± 0.5 per site for weak binding sites at 9 MHZ. The dissociation constant of Mn(II) for tight binding sites on the enzyme exhibits strong temperature dependence. In the low-temperature region (5–12 °C) brain hexokinase probably undergoes a conformational change. Frequency dependence of the normalized relaxation rate for bound water at various temperatures has shown that the number of exchangeable water molecules left in the first coordination sphere of bound Mn(II) is about one at 30 °C and about two at 18 °C. Binding of glucose 6-phosphate to hexokinase results in large-line broadening of the resonances of anomeric protons of the sugar. However, no such effect was observed in the case of glucose binding. These results suggest different modes of interaction of these two sugars to hexokinase. Line broadening of the C-(1) hydrogen resonances of glucose caused by Mn(II) in the presence of hexokinase suggests the proximity of the Mn(II) binding site to that of glucose. A lower limit of 1330 ± 170 s −1 for the rate of dissociation of glucose from enzyme-Mn(II)-glucose complex has been obtained from these studies.