Chemical Exchange Rates Research Articles

Magnesium-25 nuclear magnetic resonance in aqueous phosphate solutions

Magnesium-25 nuclear magnetic resonance is studied in aqueous solutions of molecules containing phosphate groups which bind magnesium ion. The line width of magnesium ion in 1.5 M magnesium chloride solutions is 3.8 ± 0.5 Hz and is significantly broadened by the addition of phosphate-containing molecules such as ATP. The line broadening is shown to contain effects due to the nuclear electric quadrupole interaction as well as effects due to chemical exchange of the magnesium ion between complexed and uncomplexed sites in solution. The chemical exchange rate of magnesium ion with ATP is measured to be 2 × 10° sec −1 at 25°C, an ionic strength of 4.5 and pH of 7.9. The possibility of using magnesium-25 nuclear magnetic resonance to study magnesium ion interactions with enzymes is examined.

Multi-site chemical exchange by nmr

The application of a recent multi-site chemical exchange formulation of the Bloch equations is made to an intramolecular and an intermolecular process. The experimental precautions to achieve a good complete line shape lit are discussed in some detail. The rates of internal rotation about the C—N bond in N,N-dimethylcarbamyl fluoride have been measured, and the relative signs of the long range couplings H—C—N—C—F 4J proton to fluorine are obtained from the exchange modified spectra as well as from the comparison of the 4J measured in the fast and slow exchange limits. A 4-site rate matrix is defined with one adjustable parameter for the spectral fit. The measurements are especially difficult because of the small chemical shift varying between 2.2 Hz and 1.4 Hz over the range of interest. The long range coupling methyl to fluorine are 0.8 ± 0.05 Hz trans and 0.3 ± 0.05 Hz cis and do not vary with temperature within 0.05 Hz for a 16.5 % solution in carbon tetrachioride. The intermolecular exchange of halogens in the dimethyltin dihalide mixtures is formulated in detail. A 15 site exchange process breaks down into 5 x 3 exclusive exchange groups and providing line positions and intensities are known with the line width in the absence of exchange, the whole spectrum can be reproduced using only 2-site rate matrix elements. Representative experimental and computed spectra are presented. The relationship of the matrix elements to chemical rate constants is derived but determinations of these constants are not yet complete. INTRODUCTION The study of chemical exchange rates through the modified transverse magnetization is well understood and the theory is highly developed (for example references 1—8). Some simple aspects of chemical exchange theory in nmr spectra for first order cases have been emphasized in a recent paper by Reeves and Shaw9. For intramolecular exchange in the first order limit, the determination of relative signs of coupling constants to non-exchanging nuclei influences the line shapes in a readily recognizable manner'°' Exchanges among many sites (Larmor frequencies) of arbitrary population, saturation effects and site-dependent relaxation times have been accommodated in this recent theory9. A single matrix diagonalization enables sub.tL.W.R. thanks the NRCC for financial support in the form of operating and capital equipment grants.

A line width method for determining chemical exchange rates from NMR spectra

AbstractThe applicability of W½, the line‐width at one‐half height corrected for field inhomogeneity and couplings, for characterization of nuclear magnetic resonance line shapes generated by exchange averaging of chemical shifts has been investigated for the case PA = PB. The Gutowsky‐Holm equation, simplified by the assumption of a large T2º such that T = 0, was used to produce a family of curves relating W½ corrected to the rate of exchange for various ΔvAB values.The rate of internal rotation about the CN amide bond has been studied in neat N,N‐dimethyl‐formamide between 79·5 and 159° by the W½ method and the results, Ea = 24·9 and 24·1 Kcal/mole at 60 and 100 MHz do not agree with those recently reported by Rabinovitz and Pines, Ea = 20·5 Kcal/mole obtained by the total line shape method. For neat N,N‐dimethylacetamide and for solutions of DMA in dimethylsulfoxide‐d6, application of the W½ method yielded Ea, ΔH‡ and ΔS‡ values which are in excellent agreement with those obtained by Neuman and Jonas for DMA‐dε using a total line shape analysis. The thermodynamic parameters for neat N,N‐dimethylcarbamoyl chloride obtained by the total line shape and W½ methods are also in excellent agreement, 16·4 and 16·8 Kcal/mole for Ea and 15·8 and 16·2 Kcal/mole for ΔH‡, respectively.The W½ method was also used for the determination of the activation parameters for rotation about the carbon‐nitrogen bond in the following thioamides: N,N‐dimethylthiobenzamide, (Ea = 19.8 Kcal/mole), N,N‐diethylthiobenzamide, (Ea = 20·4 Kcal/mole), N,N‐dimethylphenylthio‐acetamide (Ea = 21·4 Kcal/mole). The results obtained for these thioamides are compared with the corresponding amides. Specifically N,N‐dimethylbenzamide and N,N‐diethylbenzamide were studied using the W½ method and activation energies for rotation about the carbon‐nitrogen bond of 17·5 and 15·6 Kcal/mole, respectively, were found. The relative magnitudes of these parameters are discussed on the basis of molecular geometry of the thioamides. Attention is drawn to the fact that for structurally related amides and thioamides, the barrier heights for internal rotation are not greatly different.

Oxidized RNase as a protein model having no contribution to the hydrogen exchange rate from conformational restrictions.

As oxidized RNase is a model for the random conformation state of RNase, the hydrogen exchange kinetics of oxidized RNase approximate the intrinsic conformation-independent chemical exchange rate of the native protein. The energy of activation, the pH(min), and the k(min) of oxidized RNase exchange rates are similar to those reported for amino acid homopolymers. However, unlike the exchange from homopolymers, the exchange from oxidized RNase is characterized by a distribution of first-order rates. This distribution is important to the analysis of exchange from native proteins in terms of classes of sites which share common structural properties.

Interaction of a Spin-labeled Analogue of Nicotinamide Adenine Dinucleotide with Alcohol Dehydrogenase: III. Thermodynamic, Kinetic, and Structural Properties of Ternary Complexes as Determined by Nuclear Magnetic Resonance

Abstract ADP-C5H4(CH3)4N(·)/—O (ADP-R·) is a paramagnetic analogue of NAD in which the unpaired electron is located in a region which corresponds to the pyridine N-ribose C1 bond of the coenzyme. Studies of the effect of ADP-R· on the longitudinal nuclear relaxation rate (1/T1) of the protons of have previously shown this analogue to form binary complexes with liver alcohol dehydrogenase and to form a ternary enzyme-ADP-R·-ethanol complex. By the same technique, ternary complexes are shown here with acetaldehyde and isobutyramide. The dissociation constants of all ternary complexes are in reasonable agreement with those reported for the respective kinetically active species. All of the ternary complexes are less effective in relaxing protons than is the binary e-ADP-R· complex, suggesting that ethanol, acetaldehyde, and isobutyramide displace molecules which are hydrogen-bonded to the paramagnetic nitroxide group of enzyme-bound ADP-R·. This is directly shown by the effect of e-ADP-R· on the transverse relaxation rates (1/T2) of the protons of ethanol, acetaldehyde, and isobutyramide. In the absence of enzyme, high concentrations of ADP-R· (5 mm) increase 1/T2 of the protons of ethanol. Its effect on the methylene protons is g3 times its effect on the methyl protons. The complex of ADP-R· with liver alcohol dehydrogenase at much lower concentrations (∼0.2 mm) relaxes the protons of ethanol and the effect on the methyl protons is 8 to 23 times greater than the effect on the methylene protons, suggesting that the enzyme reorients the ethanol with reference to the unpaired electron of ADP-R· in the ternary complex. Depending on conditions, the enzyme enhances the effect of ADP-R· on 1/T2 of the methylene protons by a factor of 1.5 to 13.4 and on the methyl protons by a factor of 16 to 148. The e-ADP-R· complex is comparably effective in relaxing the protons of acetaldehyde and of isobutyramide, indicating that the unpaired electron is very near the bound substrates and inhibitor in the respective ternary complexes. All of these effects are reversed by the addition of excess NADH. The temperature dependence of 1/T2 of the methyl protons of ethanol in the presence of the enzyme-ADP-R ·-ethanol complex can be fit by two rate processes: 1/τm, the rate of chemical exchange of ethanol molecules into the ternary complex, and 1/T2m, the relaxation rate of ethanol molecules which are in the ternary complex. From 1/τm, the exchange rate of ethanol into the ternary complex is 610 sec-1 at 25° which is two orders of magnitude faster than the maximum turnover number of the alcohol dehydrogenase reaction. Lower limits of 770 sec-1 and 180 sec-1 are set on the exchange rates of acetaldehyde and isobutyramide into their ternary complexes. From the dipolar contributions to 1/T2m, the average distances between the unpaired electron of enzyme-bound ADP-R · and the protons of the bound substrates and inhibitors, calculated from the Solomon-Bloembergen equation, are: ethanol (methyl), 3.6 A; ethanol (methylene), 4.1 A; acetaldehyde (aldehyde), 3.1 A; isobutyramide (methyl), ≤3.9 A. It is concluded that substrates bind to liver alcohol dehydrogenase on the water side of the bound coenzymes and directly overlie the ribosidic bond to pyridine. Structures consistent with the calculated distances permit molecular contact between the hydrogen-donating and hydrogen-accepting positions of the substrates and the coenzymes and, therefore, support the direct transfer of hydrogen between them. Although hydrogen transfer by way of stacked pyridine and indole rings can be excluded, though the participation of tryptophan in an edge to edge structure with the pyridine ring cannot be excluded.

Electrochemically Controlled Ion Exchange: II . Transport Processes

At present, the highest current density that has resulted in the efficient removal of ions is approximately 1.5 ma/cm2. Chemical ion exchange rates were determined at ion exchange sheets prepared in a manner similar to electrode fabrication. It was concluded that transport through the binder limited the rate of exchange. A simple model was used to predict the critical current density for the process.

The Enzymatic Carboxylation of Phosphoenolpyruvate: IV. The binding of manganese and substrates by phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase

Abstract The binding of Mn2+ and substrates to phosphoenolpyruvate carboxykinase has been studied by four methods: equilibrium dialysis, gel filtration, electron paramagnetic resonance, and the proton relaxation rate of water (PRR). A binary enzyme-Mn2+ complex was detected by electron paramagnetic resonance and PRR. Binary enzyme-substrate complexes were detected for guanosine triphosphate and guanosine diphosphate by equilibrium dialysis and for inosine diphosphate by gel filtration. Ternary complexes containing enzyme, Mn2+, and substrate were detected for IDP by gel filtration and PRR, for phosphoenolpyruvate by equilibrium dialysis and PRR, and for inosine triphosphate and oxalacetate by PRR. Higher order complexes of the form eMn-sMn were detected for GDP and GTP by equilibrium dialysis. Phospholactate, an analogue of phosphoenolpyruvate, formed ternary complexes with enzyme and Mn2+ and a stable quaternary complex with enzyme, Mn2+, and IDP, as detected by PRR. Where comparable, the dissociation constants obtained by the various methods were in good agreement. The enzyme enhanced the effect of Mn2+ on the PRR of water (eb = 14.2), and this enhancement was reduced in the ternary complexes with phosphoenolpyruvate (et = 6.7) and with IDP (et = 10.4). Since the relaxation mechanism was the same for each of these complexes (measuring the rate of chemical exchange of water protons into the coordination sphere of Mn2+), the relative values of e are consistent with the formation of enzyme-metal-substrate bridge complexes. The related enzyme, phosphoenolpyruvate carboxylase, gives similar results, forming a binary enzyme-Mn2+ complex (eb = 13.8) and a ternary complex with phosphoenolpyruvate (et ≤ 11.3). The Kd of the binary complex agrees with the activator constant for Mn2+ obtained kinetically. The complexes formed by both carboxylating enzymes are thus similar to those found previously for muscle pyruvate kinase and, therefore, suggest homologous mechanisms for phosphoenolpyruvate carboxykinase, phosphoenolpyruvate carboxylase, and pyruvate kinase.

Comparison of chemical exchange rates determined by nuclear magnetic resonance line-shape and equilibration methods. Internal rotation of N-methyl-N-benzylformamide

Comparison of chemical exchange rates determined by nuclear magnetic resonance line-shape and equilibration methods. Internal rotation of N-methyl-N-benzylformamide

Exchange Rates by Nuclear Magnetic Multiple Resonance. III. Exchange Reactions in Systems with Several Nonequivalent Sites

The nuclear magnetic multiple resonance method for the study of chemical exchange rates has been applied to systems in which a nucleus X is reversibly exchanged between three nonequivalent sites. A procedure is outlined, which is applicable to systems with any number of sites and permits the determination of the lifetime τK at any one site K. In this procedure the effective ``relaxation time'' τ1K and the equilibrium z magnetization, M0K (τ1K/T1K), are determined in experiments (Type I) in which the X signals at all other sites are completely saturated. The rate constants λKL and λLK of the individual exchange reactions (K⇄L) may then be determined in Type II experiments in which the signals of all sites excluding K and L are saturated. In Type II experiments only the equilibrium signal intensities need be measured. The method has been applied to the base-catalyzed proton exchange in the keto—enol system of acetylacetone and it was found that exchange between the olefinic =CH and hydroxylic —OH protons occurs merely as a consequence of the keto—enol transformation. Within the accuracy of the measurements no evidence was found for a proton exchange between the =CH and —OH protons which does not involve the CH2 group in the keto form as an intermediate.

Study of Moderately Rapid Chemical Exchange Reactions by Means of Nuclear Magnetic Double Resonance

A nuclear magnetic double-resonance method for the determination of chemical exchange rates has been developed. The method is applicable to systems in which a nuclear spin is reversibly transferred between two nonequivalent sites, A and B. The lifetime (τA) and spin—lattice relaxation time (T1A) in Site A are obtained through the study of the decay to a new equilibrium value of Signal A upon the sudden saturation of Signal B. The converse experiment permits the determination of τB and T1B. A number of data for cross checks are furthermore obtained through the study of the recovery of the signals upon the release of various combinations of saturating rf fields. A simple theory based on the Bloch equations as modified by McConnell to incorporate the effects of chemical exchange is given. Experimental results on the hydroxyl proton exchange in the system salicylaldehyde and 2-hydroxyacetophenone are well described by this simple theory. The present method, which can readily be extended to systems with several sites, offers a complement to the Gutowsky—Saika single-resonance method and is particularly suited to the study of exchange rates slower than those accessible by the single-resonance method.