Measured Equilibrium Constants Research Articles

ChemInform Abstract: High Pressure Mass Spectrometric Studies of Silicon-Containing Ions

Association reactions of ions with neutral molecules are readily studied using HPMS. These equilibrium reactions are studied with reactants and products in thermal equilibrium at well defined and measurable temperatures and pressures. The standard enthalpy and entropy changes for the association reactions are obtainable from the variation of the measured equilibrium constants with temperature. This thermodynamic data may be used to obtain other data such as proton affinities and stabilization energies. The quality of this derived information for organosilicon compounds is limited at present, not by the HPMS data but by the required ancillary data, for example the standard enthalpies of formation of neutral organosilicon compounds.

ChemInform Abstract: Thermodynamics for the Hydrogenation of Dimanganese Decacarbonyl.

The equilibrium constant for the hydrogenation of Mn{sub 2}(CO){sub 10} in supercritical carbon dioxide has been measured under carbon monoxide and hydrogen at total system pressures of 142-300 atm by in situ {sup 55}Mn and {sup 1}H NMR spectroscopy over the temperature range 165-220C. The hydrogenation of Mn{sub 2}(CO){sub 10} is efficiently promoted by the addition of Co{sub 2}(CO){sub 8} to the system. The rate of HMn(CO){sub 5} production at 100C is increased by 2 orders of magnitude with the addition of 1 equiv of Co{sub 2}(CO){sub 8}. The magnitude of this promotional effect has extended the range of temperatures down to 80C over which it is feasible to determine the equilibrium constant for Mn{sub 2}(CO){sub 10} hydrogenation. Van't Hoff plots for the hydrogenation of Mn{sub 2}(CO){sub 10} in the presence and absence of the Co{sub 2}(CO){sub 8} promoter were found to yield standard enthalpy and entropy changes that agree to within the statistical error limit of the equilibrium constant measurements. The resultant analysis for the combined data set yields {Delta}H{degree} = 8.7 {plus minus} 0.3 kcal/mol and {Delta}S{degree} = 8.5 {plus minus} 0.8 cal/(K{center dot}mol) for the hydrogenation of Mn{sub 2}(CO){sub 10}. The heterobimetallic dimer Mn(CO){sub 5}-Co(CO){sub 4} ismore » observed as an additional reaction product in both the {sup 55}Mn and the {sup 59}Co NMR spectra, within the mixed-metal system. The thermodynamics for the redistribution reaction yielding 2 equiv of MnCo(CO){sub 9} from the homonuclear dimers, Mn{sub 2}(CO){sub 10} and Co{sub 2}(CO){sub 8} was found to be nearly thermal neutral exhibiting {Delta}H{degree} = 0.8 {plus minus} 0.3 kcal/mol and {Delta}S{degree} = 0.7 {plus minus} 0.8 cal/(K{center dot}mol), respectively.« less

Excited State Lactim to Lactam Type Tautomerization Reaction in 5-(4-Fluorophenyl)-2-Hydroxypyridine: Spectroscopic Study and Quantum Chemical Calculation

The photophysical properties of 5-(4-fluorophenyl)-2-hydroxypyridine (FP2HP) have been studied by steady state and time resolved spectroscopy in combination with quantum chemical calculations. The molecule FP2HP exists as lactim and lactam form in the ground state due to small stability difference but does not undergo lactim to lactam isomerisation due to high barrier energy. Whereas in the excited state the lactim form undergoes tautomerization producing red shifted emission of the lactam tautomer along with the local emission of the lactim form. In polar protic solvents, the barrier for lactim-lactam tautomerization rapidly decreases forming the lactam tautomer only. Temperature has pronounced effect on the excited state tautomerization equilibrium and is clearly reflected in the measured equilibrium constant (K(tau)(0)) and free energy change (ΔG(0)). Structural calculations at Hartree Fock and Density Functional Theory levels, calculated stability of the isomers in different solvents using Polarized Continuum Model and the theoretical potential energy surfaces for the ground and excited states along the proton transfer coordinate are reported for the tautomeric equilibrium of FP2HP.

Generation and Characterization of 1,2-Diaryl-1,1,2,2-tetramethyldisilane Cation Radicals

Nanosecond laser flash photolysis methods were used to generate and spectrally characterize the cation radicals of 1,2-diaryl-1,1,2,2,-tetramethyldisilanes (Ar = p-X-Ph, X = H, CH(3), OCH(3)) in hexafluoroisopropanol (HFIP) at room temperature. The disilane cation radicals rapidly reacted with methanol, with bimolecular rate constants ranging from 0.63 to 2.1 x 10(8) M(-1) s(-1). The cation radicals were found to react with tert-butanol 4-5 times more slowly than methanol, consistent with a small steric effect for nucleophile-assisted fragmentation of the Si-Si bond. The standard potentials for oxidation of the disilanes in HFIP were determined by two different methods: first, by measuring equilibrium constants for electron exchange between the disilanes and the cation radical of hexaethylbenzene and, second, by combining electrochemical data from cyclic voltammetry with the lifetimes of the disilane cation radicals measured by laser flash photolysis in the same media. Agreement between the two methods was excellent (<or=3 mV). The oxidation of 1,2-di-p-methoxyphenyl-1,1,2,2,-tetramethyldisilane by slow scan cyclic voltammetry in acetonitrile was not found to be reversible, in contrast to prior literature reports. Possible explanations for the prior results are proposed.

A thermodynamic study of selective solvation in solvent mixtures

Changes in the (31)P NMR chemical shift of tri-n-butylphosphine oxide have been measured as function of solvent composition in a number of binary solvent mixtures. The data were analysed using a model that separates the contributions of specific H-bond interactions with the first solvation shell and the non-specific effects of the bulk solvent on the chemical shift. This allowed measurement of equilibrium constants between differently solvated states of the probe and hence thermodynamic quantification of preferential solvation in the binary mixtures. The results are analysed in the context of the electrostatic solvent competition model, which assumes that solvent effects on intermolecular interactions can be interpreted based on the exchange of specific functional group contacts, with minimal involvement of the bulk solvent. The thermodynamic measurements of preferential solvation were used to determine the H-bond donor parameter alpha for cyclohexane, n-octane, n-dodecane, benzene, 1,4-dioxane, carbon tetrachloride, acetone, dichloromethane, dimethyl sulfoxide and chloroform. For solvents where the H-bond donor parameters have been measured as solutes in carbon tetrachloride solution, the H-bond donor parameters measured here for the same compounds as solvents are practically identical, i.e. solute and solvent H-bond parameters are directly interchangable. For alkanes, the experimental H-bond donor parameter is significantly larger than expected based on calculated molecular electrostatic potential surfaces. This might suggest an increase in the relative importance of van der Waals interactions when electrostatic effects are weak.

Kinetic and thermochemical studies of the ClO + ClO + M ⇄ Cl2O2 + M reaction

Recent work by von Hobe et al. [Atmos. Chem. Phys., 2007, 7, 3055] has highlighted significant inconsistencies between laboratory results, theoretical calculations and field observations concerning the ClO dimer ozone destruction cycle. This work investigates the temperature dependence of the equilibrium constant of one of the key reactions in this cycle, ClO + ClO + M <=> Cl(2)O(2) + M (1, -1), by means of laser flash photolysis and time-resolved UV absorption spectroscopy. ClO radicals were generated via laser flash photolysis of Cl(2)/Cl(2)O mixtures in synthetic air. Radicals were monitored via UV absorption spectroscopy: the use of a charge coupled device (CCD) detector allowed time resolution over a broad range of wavelengths giving unequivocal concentrations of radicals. The equilibrium constant K(eq) was determined as the ratio of the rate constants of the forward and reverse over the temperature range T = 256.55-312.65 K. Second Law and Third Law thermodynamic methods were employed to determine the standard enthalpy and entropy changes of , Δ(r)H° and Δ(r)S°, from the measured equilibrium constants. The values obtained from Second Law analysis were Δ(r)H° = - 80.7 ± 2.2 kJ mol(-1) and Δ(r)S° = -168.1 ± 7.8 J K(-1) mol(-1). Third Law analysis gave Δ(r)H° = -74.65 ± 0.4 kJ mol(-1) and Δ(r)S° = -148.0 ± 0.4 J K(-1) mol(-1). These values are in good agreement with previous work by Nickolaisen et al. [J. Phys. Chem., 1994, 98, 155] but greater in (negative) magnitude than current JPL-NASA recommendations [Sander et al., Chemical Kinetics and Photochemical Data for Use in Atmospheric Studies, JPL Publication 06-2, NASA Jet Propulsion Laboratory, Pasadena, 2006 (interim update to this reference, 2009)]. The discrepancy between the Second and Third Law analyses also agrees with Nickolaisen et al., possibly indicating an aspect of the ClO recombination reaction not yet fully elucidated. The atmospheric implications of the results and their impact on the current understanding on polar ozone depletion are briefly discussed.

Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes

The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca(2+)-pump (PMCA) in the membrane region upon interaction with Ca(2+), calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16, measuring the shift of conformation E(2) to the auto-inhibited conformation E(1)I and to the activated E(1)A state, titrating the effect of Ca(2+) under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca(2+) regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca(2+) transport but does not modify the affinity for Ca(2+) at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E(1)I to the activated E(1)A conformation and thus modulating the transport of Ca(2+). This is reflected in the different apparent constants for Ca(2+) in the absence of CaM (calculated by Ca(2+)-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca(2+) site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca(2+) and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca(2+) binding and activation.

Quantitative assignment of reaction directionality in constraint-based models of metabolism: Application to Escherichia coli

Constraint-based modeling is an approach for quantitative prediction of net reaction flux in genome-scale biochemical networks. In vivo, the second law of thermodynamics requires that net macroscopic flux be forward, when the transformed reaction Gibbs energy is negative. We calculate the latter by using (i) group contribution estimates of metabolite species Gibbs energy, combined with (ii) experimentally measured equilibrium constants. In an application to a genome-scale stoichiometric model of Escherichia coli metabolism, iAF1260, we demonstrate that quantitative prediction of reaction directionality is increased in scope and accuracy by integration of both data sources, transformed appropriately to in vivo pH, temperature and ionic strength. Comparison of quantitative versus qualitative assignment of reaction directionality in iAF1260, assuming an accommodating reactant concentration range of 0.02–20 mM, revealed that quantitative assignment leads to a low false positive, but high false negative, prediction of effectively irreversible reactions. The latter is partly due to the uncertainty associated with group contribution estimates. We also uncovered evidence that the high intracellular concentration of glutamate in E. coli may be essential to direct otherwise thermodynamically unfavorable essential reactions, such as the leucine transaminase reaction, in an anabolic direction.

Aromatic Residues ∈Trp-55 and δTrp-57 and the Activation of Acetylcholine Receptor Channels

The two transmitter binding sites of the neuromuscular acetylcholine (ACh) receptor channel contain several aromatic residues, including a tryptophan located on the complementary, negative face of each binding pocket. These two residues, Trp-55 in the epsilon subunit and Trp-57 in the delta subunit, were mutated (AEFHILRVY), and for most constructs the rate constants for acetylcholine binding and channel gating were estimated by using single channel kinetic analyses. The rate constants for unliganded channel opening and closing were also estimated for some mutants. From these measurements we calculated all of the equilibrium constants of the "allosteric" cycle as follows: diliganded gating, unliganded gating, dissociation from the C(losed) conformation, and dissociation from the O(pen) conformation. The results indicate the following. (i) These aromatic side chains play a relatively minor role in ACh receptor channel activation. (ii) The main consequence of mutations is to reduce the affinity of the O conformation of the binding site for ACh, with the effect being greater at the epsilon subunit. (iii) In epsilon (but not delta) the aromatic nature of the side chain is important in determining affinity, to a slightly greater degree in the O conformation. Phi value analyses (of both tryptophan residues) show Phi approximately 1 for both the ACh binding and diliganded gating reactions. (iv) This suggests that the structural boundaries of the dynamic elements of the gating conformational change may not be subunit-delimited, and (v) the mutated tryptophan residues experience energy changes that occur relatively early in both the ligand-binding and channel-gating reactions.

Reversible Oxidative Addition and Reductive Elimination of Fluorinated Disulfides at Gold(I) Thiolate Complexes: A New Ligand Exchange Mechanism

Reaction of HAuCl4 x 3 H2O with excess HSAr (Ar = C6F5 or C6F4H) in ethanol, followed by addition of [Et4N]Cl, produced [Et4N][Au(SAr)4] (Ar = C6F5 (1a) or C6F4H (1b)) as red crystalline solids in high yield. These complexes are rare examples of homoleptic gold(III) thiolate complexes. The crystal structures 1 show square planar geometry at the gold center with elongated Au-S bonds. Both complexes undergo reversible reductive elimination/oxidative addition processes in solution via thermal and photochemical pathways. Equilibrium constant and photostationary state measurements indicate that the relative importance of the two pathways depends on the nature of the aromatic groups. The metal-containing reductive elimination products, [Et4N][Au(SAr)2] (Ar = C6F5 (2a) or C6F4H (2b)), were confirmed by both independent synthesis and crystallographic characterization. Cross-reactions between either 1 or 2 and various disulfides led to ligand exchange via an addition-elimination process, a previously unknown reaction pathway for ligand exchange at gold(I) centers.

Differential Selectivity of the Escherichia coli Cell Membrane Shifts the Equilibrium for the Enzyme-Catalyzed Isomerization of Galactose to Tagatose

An Escherichia coli galactose kinase gene knockout (DeltagalK) strain, which contains the l-arabinose isomerase gene (araA) to isomerize d-galactose to d-tagatose, showed a high conversion yield of tagatose compared with the original galK strain because galactose was not metabolized by endogenous galactose kinase. In whole cells of the DeltagalK strain, the isomerase-catalyzed reaction exhibited an equilibrium shift toward tagatose, producing a tagatose fraction of 68% at 37 degrees C, whereas the purified l-arabinose isomerase gave a tagatose equilibrium fraction of 36%. These equilibrium fractions are close to those predicted from the measured equilibrium constants of the isomerization reaction catalyzed in whole cells and by the purified enzyme. The equilibrium shift in these cells resulted from the higher uptake and lower release rates for galactose, which is a common sugar substrate, than for tagatose, which is a rare sugar product. A DeltamglB mutant had decreased uptake rates for galactose and tagatose, indicating that a methylgalactoside transport system, MglABC, is the primary contributing transporter for the sugars. In the present study, whole-cell conversion using differential selectivity of the cell membrane was proposed as a method for shifting the equilibrium in sugar isomerization reactions.

Thermodynamic properties of lanthanum and lanthanide halides: IV. Enthalpies of atomization of LnCl, LnCl+, LnF, LnF+, and LnF2

The results of measurement of equilibrium constants of 30 reactions involving lanthanum and lanthanide fluorides (LnF, LnF2, and LnF3) and 14 reactions involving lanthanum and lanthanide monochlorides (Ln = La-Lu) have been summarized. These constants have been used for calculating the enthalpies of reactions by the second and third laws, from which the enthalpies of atomization ΔatH00 of LnCl, LnF, and LnF2 have been determined. Comparison of the calculation results shows that the thermodynamic functions of LnCl and LnF (Ln = Ce-Yb) in which the electronic excitation contribution has been calculated from the excitation energies of Ln+ ions allow one to adequately determined the ΔatH00 values from experimental data. Using the trends in the change in ΔatH00 as a function of the atomic number of a lanthanide, the enthalpies of atomization of compounds for which experimental data are lacking have been estimated. The ΔatH00 values for LnCl+ ions have been calculated. The reliability of the ΔatH00 values for LnF+ ions have been assessed.

Restricted Active Site Docking by Enzyme-bound Substrate Enforces the Ordered Cleavage of Prothrombin by Prothrombinase

The preferred pathway for prothrombin activation by prothrombinase involves initial cleavage at Arg(320) to produce meizothrombin, which is then cleaved at Arg(271) to liberate thrombin. Exosite binding drives substrate affinity and is independent of the bond being cleaved. The pathway for cleavage is determined by large differences in V(max) for cleavage at the two sites within intact prothrombin. By fluorescence binding studies in the absence of catalysis, we have assessed the ability of the individual cleavage sites to engage the active site of Xa within prothrombinase at equilibrium. Using a panel of recombinant cleavage site mutants, we show that in intact prothrombin, the Arg(320) site effectively engages the active site in a 1:1 interaction between substrate and enzyme. In contrast, the Arg(271) site binds to the active site poorly in an interaction that is approximately 600-fold weaker. Perceived substrate affinity is independent of active site engagement by either cleavage site. We further show that prior cleavage at the 320 site or the stabilization of the uncleaved zymogen in a proteinase-like state facilitates efficient docking of Arg(271) at the active site of prothrombinase. Therefore, we establish direct relationships between docking of either cleavage site at the active site of the catalyst, the V(max) for cleavage at that site, substrate conformation, and the resulting pathway for prothrombin cleavage. Exosite tethering of the substrate in either the zymogen or proteinase conformation dictates which cleavage site can engage the active site of the catalyst and enforces the sequential cleavage of prothrombin by prothrombinase.

Magnesium dependence of the measured equilibrium constants of aminoacyl-tRNA synthetases

The apparent equilibrium constants ( K′) for six reactions catalyzed by aminoacyl-tRNA synthetases from Escherichia coli were measured, the equations for the magnesium dependence of the equilibrium constants were derived, and best-fit analyses between the measured and calculated values were used. The K′ values at 1 mM Mg 2+ ranged from 0.49 to 1.13. The apparent equilibrium constants increased with increasing Mg 2+ concentrations. The values were 2–3 times higher at 20 mM Mg 2+ than at 1 mM Mg 2+, and the dependence was similar in the class I and class II synthetases. The main reason for the Mg 2+ dependence is the existence of PP i as two magnesium complexes, but only one of them is the real product. AMP exists either as free AMP or as MgAMP, and therefore also has some effect on the measured equilibrium constant. However, these dependences alone cannot explain the measured results. The measured dependence of the K′ on the Mg 2+ concentration is weaker than that caused by PP i and AMP. Different bindings of the Mg 2+ ions to the substrate tRNA and product aminoacyl-tRNA can explain this observation. The best-fit analysis suggests that tRNA reacts as a magnesium complex in the forward aminoacylation direction but this given Mg 2+ ion is not bound to aminoacyl-tRNA at the start of the reverse reaction. Thus Mg 2+ ions seem to have an active catalytic role, not only in the activation of the amino acid, but in the posttransfer steps of the aminoacyl-tRNA synthetase reaction, too.

Study of Bacteriophage T4-encoded Dam DNA (Adenine-N6)-methyltransferase Binding with Substrates by Rapid Laser UV Cross-linking

DNA methyltransferases of the Dam family (including bacteriophage T4-encoded Dam DNA (adenine-N(6))-methyltransferase (T4Dam)) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. In this study, we describe the application of direct (i.e. no exogenous cross-linking reagents) laser UV cross-linking as a universal non-perturbing approach for studying the characteristics of T4Dam binding with substrates in the equilibrium and transient modes of interaction. UV irradiation of the enzyme.substrate complexes using an Nd(3+):yttrium aluminum garnet laser at 266 nm resulted in up to 3 and >15% yields of direct T4Dam cross-linking to DNA and AdoMet, respectively. Consequently, we were able to measure equilibrium constants and dissociation rates for enzyme.substrate complexes. In particular, we demonstrate that both reaction substrates, specific DNA and AdoMet (or product AdoHcy), stabilized the ternary complex. The improved substrate affinity for the enzyme in the ternary complex significantly reduced dissociation rates (up to 2 orders of magnitude). Several of the parameters obtained (such as dissociation rate constants for the binary T4Dam.AdoMet complex and for enzyme complexes with a nonfluorescent hemimethylated DNA duplex) were previously inaccessible by other means. However, where possible, the results of laser UV cross-linking were compared with those of fluorescence analysis. Our study suggests that rapid laser UV cross-linking efficiently complements standard DNA methyltransferase-related tools and is a method of choice to probe enzyme-substrate interactions in cases in which data cannot be acquired by other means.

Two Methods for the Determination of Enantiomeric Excess and Concentration of a Chiral Sample with a Single Spectroscopic Measurement

The previously established enantioselective indicator-displacement assays (eIDAs) for the determination of concentration and enantiomeric excess (ee) require two spectroscopic measurements for each chiral sample. To further simplify the operation of eIDAs, we now introduce two innovative analytical methods, both of which utilize a dual-chamber quartz cuvette, which reduces the number of spectroscopic measurements from two to one. An attractive feature of this cuvette is that the concentration- and ee-dependent absorption data can be collected at the isosbestic points or transparent regions of the spectra recorded in each individual chamber, thereby reflecting optical changes that occur in the other chamber. Therefore, two independent equations, which are needed to solve the values of the two independent variables-concentration and ee-can be established with only a single spectroscopic measurement. The first method takes advantage of this feature in conjunction with a judicious choice of indicator/host combinations to generate concentration- and ee-dependent calibration curves. Our second method removes the requirement to measure equilibrium constants and molar absorptivities altogether through the use of artificial neural networks (ANNs). The most frequently used three-layer feed-forward network is generated, which relates the absorption data to concentration and ee of the samples by training with a back propagation procedure. Here, the data collection is not limited to the isosbestic points or transparent regions. Both approaches enabled accurate and rapid determination of concentration and ee of chiral samples. The technology removes the relative difficulty, which is the need for two separate measurements for concentration and ee respectively, of analyzing chiral samples compared to achiral samples. When implemented in a high-throughput format, this technology should greatly facilitate the discovery of asymmetric catalysts in the same way as conventional high-throughput screening assays.

Hydrogen-Bond Interactions of Nicotine and Acetylcholine Salts: A Combined Crystallographic, Spectroscopic, Thermodynamic and Theoretical Study

The hydrogen-bond (HB) interactions of the monocharged active forms of nicotine and acetylcholine (ACh) have been compared theoretically by using density functional theory (DFT) calculations and experimentally on the basis of crystallographic observations and the measurement of equilibrium constants in solution. The 2,4,6-trinitrophenolate (picrate) counterion was used to determine the experimental HB basicity of the cations despite its potential multisite HB acceptor properties. The preferred HB interaction site of the ammonium picrate salts was determined from a survey of crystallographic data found in the Cambridge Structural Database (CSD) and is supported by theoretical calculations. Two distinct classes of ammonium groups were characterised depending on the absence (quaternary ammonium) or presence (tertiary, secondary and primary ammoniums) of an N(+)HO hydrogen bond linking the two ions. The crystal structure of nicotinium picrate was determined and compared with that of ACh. This analysis revealed the peculiar behaviour of the ammonium moiety of nicotinic acetylcholine receptor (nAChR) ligands towards the picrate anion. Dedicated methods have been developed to separate the individual contributions of the anion and cation accepting sites to the overall HB basicity of the ion pairs measured in solution. The HB basicities of the picrate anions associated with the two different ammonium classes were determined in dichloromethane solution by using several model ion pairs with non-basic ammonium cations. The experimental and theoretical studies performed on the nicotine and ACh cations consistently show the significant HB ability of the acceptor site of nAChR agonists in their charged form. Both the greater HB basicity of the pyridinic nitrogen over the carbonyl oxygen and the greater HB acidity of the N(+)H unit relative to N(+)CH could contribute to the higher affinity for nAChRs of nicotine-like ligands relative to ACh-like ligands.

Transient State Kinetics of Transcription Elongation by T7 RNA Polymerase

The single subunit DNA-dependent RNA polymerase (RNAP) from bacteriophage T7 catalyzes both promoter-dependent transcription initiation and promoter-independent elongation. Using a promoter-free substrate, we have dissected the kinetic pathway of single nucleotide incorporation during elongation. We show that T7 RNAP undergoes a slow conformational change (0.01-0.03 s(-1)) to form an elongation competent complex with the promoter-free substrate (dissociation constant (Kd) of 96 nM). The complex binds to a correct NTP (Kd of 80 microM) and incorporates the nucleoside monophosphate (NMP) into RNA primer very efficiently (220 s(-1) at 25 degrees C). An overall free energy change (-5.5 kcal/mol) and internal free energy change (-3.7 kcal/mol) of single NMP incorporation was calculated from the measured equilibrium constants. In the presence of inorganic pyrophosphate (PPi), the elongation complex catalyzes the reverse pyrophosphorolysis reaction at a maximum rate of 0.8 s(-1) with PPi Kd of 1.2 mM. Several experiments were designed to investigate the rate-limiting step in the pathway of single nucleotide addition. Acid-quench and pulse-chase kinetics indicated that an isomerization step before chemistry is rate-limiting. The very similar rate constants of sequential incorporation of two nucleotides indicated that the steps after chemistry are fast. Based on available data, we propose that the preinsertion to insertion isomerization of NTP observed in the crystallographic studies of T7 RNAP is a likely candidate for the rate-limiting step. The studies here provide a kinetic framework to investigate structure-function and fidelity of RNA synthesis and to further explore the role of the conformational change in nucleotide selection during RNA synthesis.

13C NMR measurements of acid-base equilibrium constants in alkaline aqueous solutions of D-glucose anomers

The concentration dissociation constants pKc of the α and β D-glucose anomers were measured at pH 12–14 by the 13C NMR method. The pKc values for L ⇔ H−1L + H equilibria at 25°C and an ionic strength of 1.0 mol/l (NaCl) were pKcα = 12.31 ± 0.03 and pKcβ = 12.03 ± 0.01. Dissociation was found to shift all the spectrum lines downfield. The most probable hydroxyl group responsible for the dissociation of β-D-glucose was determined.

Evaluation of the protonation thermochemistry obtained by the extended kinetic method

An evaluation of the results obtained by the extended kinetic method for a series of representative bases is presented here. Analysis of the original experimental data is conducted using the orthogonal distance regression (ODR) statistical treatment. A comparison with the proton affinities and protonation entropies obtained from variable temperature equilibrium constant measurements demonstrate deviations, which may be ascribed to random and systematic errors. Considerable random errors are associated with the extended kinetic method if the number of reference bases and the range of effective temperatures are too low. It is also confirmed that large systematic errors on proton affinities and protonation entropies are obtained when large protonation entropy is associated with the considered system. It is, however, encouraging to note that the gas phase basicities obtained by the extended kinetic method are generally comparable to that obtained by other methods within a few kJ/mol.