Fast and precise access to enantiomerization rate constants in dynamic chromatography

An analytical solution of the unified equation to evaluate elution profiles of interconverting enantiomers in dynamic chromatography is presented. Rate constants k1 and k(-1) and Gibbs activation energies are directly obtained from the chromatographic parameters (retention times tR A and tR A of the interconverting enantiomers, the peak widths at half height wA and wB, and the relative plateau height hp), and the initial amounts A0 and B0 of the enantiomers without any iterative and time consuming computational step. Therefore, this equation is no longer limited to racemic analytes. The analytical solution presented here was validated by comparison with a dataset of 125,000 simulated elution profiles of enantiomerizations. Furthermore, it was found that the recovery rate from a defined dataset is on average 40% higher using the unified equation compared to evaluation methods based on iterative computer simulation. The new equation was applied to determine the enantiomerization rate constant of 1-n-butyl-2-tert-butyldiaziridine by enantioselective gas chromatography. The activation parameters (DeltaH(double dagger) = 112.6 +/- 2.5 kJ/mol and DeltaS(double dagger) = -27 +/- 2 J/(K mol) were obtained from temperature-dependent measurements between 100 degrees C and 140 degrees C in 10K steps.     

Purification, kinetic and thermodynamic studies of a new ribonuclease from a mutant of Aspergillus niger

Ribonuclease was purified from Aspergillus niger SA-13-20 to homogeneity level by using (NH(4))(2)SO(4) precipitation, DEAE-cellulose anion-exchange chromatography, ultrafiltration and Sephacryl HR-200 chromatography. The molecular weight and isoelectric point of the enzyme was 40.1kDa and 5.3, respectively. The pH- and temperature-dependent kinetic parameters were determined. The RNase showed the strongest affinity with RNA as the substrate, and the highest catalytic efficiency for hydrolysis of the substrate at pH 3.5 and 65 degrees C. It exhibited Michaelis-Menten Kinetics with k(cat) of 118.1s(-1) and K(m) of 57.0 microg ml(-1), respectively. Thermodynamic parameters for catalysis and thermal denaturation were also determined. Activation energy (E(a)) for catalysis of A. niger SA-13-20 RNase was 50.31 kJ mol(-1) and free energy (DeltaG(#)), enthalpy (DeltaH(#)) and entropy (DeltaS(#)) of activation for catalysis of the enzyme at 65 degrees C were 69.76, 47.50 and -65.83 Jmol(-1)K(-1), respectively. Activation energy (E(a,d)) for denaturation of the enzyme was 200.53 kJ mol(-1) and free energy (DeltaG(d)(#)), enthalpy (DeltaH(d)(#)) and entropy (DeltaS(d)(#)) of activation for denaturation of the enzyme at 45 degrees C were 79.18 kJ mol(-1), 197.88 and 373.09 Jmol(-1)K(-1), respectively.     

Thermodynamics of carbohydrate isomerization reactions. The conversion of aqueous allose to psicose

The thermodynamics of the conversion of aqueous D-psicose to D-allose has been investigated using high-pressure liquid chromatography. The reaction was carried out in phosphate buffer at pH 7.4 over the temperature range 317.25-349.25 K. The following results are obtained for the conversion process at 298.15 K: DeltaG degrees = - 1.41 +/- 0.09 kJ mol(-1), DeltaH degrees = 7.42 +/- 1.7 kJ mol(-1), and DeltaC(p) degrees = 67 +/- 50 J mol(-1) K(-1). An approximate equilibrium constant of 0.30 is obtained at 333.15 K for the conversion of aqueous D-psicose to D-altrose. Available thermodynamic data for isomerization reactions involving aldohexoses and aldopentoses are summarized.     

Breakdown kinetics of the tri-chromium(III) oxo acetate cluster ([Cr(3)O(OAc)(6)]+) with some ligands of biological interest

Kinetics for the breakdown of the trinuclear chromium acetate cluster, [Cr(3)O(OAc)(6)](+), with a series of monoprotic and diprotic ligands in weakly acidic aqueous media (pH approximately 4 or approximately 5) have been investigated spectrophotometrically at 40-60 degrees C. The results point to an ion-pair equilibrium as the first step followed by associative interchange mechanism forming the mononuclear product of the reaction. Pseudo-first-order rates were determined from absorbance data and associated activation parameters were calculated using the Eyring equation. Enthalpy and entropy terms of the reactions (e.g., histidine, DeltaH(double dagger) = 75 +/- 15 kJ mol(-1), DeltaS(double dagger) = -130 +/- 25 J K(-1) mol(-1); lactic acid, DeltaH(double dagger) = 66 +/- 13 kJ mol(-1), DeltaS(double dagger) = -155 +/- 30 J K(-1) mol(-1); glycine, DeltaH(double dagger) = 31 +/- 6 kJ mol(-1), DeltaS(double dagger) = -225 +/- 45 J K(-1) mol(-1)) are consistent with an associative interchange (I(a)) mechanism, and produce a linear isokinetic plot (slope = 50 degrees C). Rates and activation parameters are comparable to those of substitution reactions of the chromium(III) hexaaqua cation. Other ligands studied included malonic acid and the amino acid, aspartic acid. Observed rates are faster than water exchange rates, but typically slower than anion substitution rates, and indicate that trinuclear chromium(III) clusters are expected to be kinetically stable in neutral to slightly acidic conditions.     

Kinetics and mechanism of the decomposition of S-nitrosoglutathione by l-ascorbic acid and copper ions in aqueous solution to produce nitric oxide.

S-Nitrosothiols serve as a good source of nitric oxide ((*)NO) mainly due to the ease of cleavage of the S-N bond which consequently produces (*)NO. The reductive decomposition of S-nitrosoglutathione (GSNO) by l-ascorbic acid (vitamin C) yields (*)NO which was monitored both electrochemically (using NO-probe) and spectrophotometrically. The rate of reaction and (*)NO release was found to be pH dependent in a manner which drastically increases with pH demonstrating that the l-ascorbic acid dianion (A(2-)) is by far the most reactive species of l-ascorbic acid (H(2)A). The derived rate expression (measuring the disappearance of the absorption at ca. 336 nm due to GSNO) was established as rate = -d[GSNO](t)/dt = ((k(a)[H(+)](2) + k(b)[H(+)]K(1) + k(c)K(1)K(2))/([H(+)](2) + K(1)[H(+)] + K(1)K(2)))[GSNO](t)[H(2)A](t). k(a), k(b), and k(c) are second-order rate constants via the H(2)A, HA(-), and A(2-) pathways, respectively, while K(1) and K(2) represent the first and second equilibrium dissociation constants of l-ascorbic acid. There is little or no reaction at low pH (below 5.5), where H(2)A is a predominant species, and as a result the rate constant (k(a)) via this route was found to be negligible. At 25 degrees C, k(b) = 5.23 +/- 1.47 x 10(-3) dm(3) mol(-1) s(-1) and k(c) = 1.22 +/- 0.04 x 10(3) dm(3) mol(-1) s(-1), activation parameters DeltaH(double dagger)(b) = 54.4 +/- 4.3 kJ mol(-1), DeltaS(double dagger)(b) = -106 +/- 16 J K(-1) mol(-1), DeltaH(double dagger)(c) = 80.5 +/- 7.5 kJ mol(-1), DeltaS(double dagger)(c) = 84 +/- 7 kJ mol(-1). The experimental rate and activation parameters suggest that this redox process follows an outer-sphere electron transfer mechanism. GSNO is relatively stable in the dark, aqueous medium and even in the presence of trace quantities of Cu(2+). Induced catalytic decomposition of GSNO only becomes significant above ca. 10 microM Cu(2+), but after this it shows linear dependency. To nullify any catalysis by Cu(2+) or any other transition metal ions, EDTA was added to all experimental reactions except those where catalysis by Cu(2+) was studied.     

Positive heat capacity change upon specific binding of translation initiation factor eIF4E to mRNA 5' cap

Specific recognition of the mRNA 5' cap by eukaryotic initiation factor eIF4E is a rate-limiting step in the translation initiation. Fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry were used to examine the thermodynamics of eIF4E binding to a cap-analogue, 7-methylGpppG. A van't Hoff plot revealed nonlinearity characterized by an unexpected, large positive molar heat capacity change (DeltaC(degree)(p) = +1.92 +/- 0.93 kJ.mol(-1).K(-1)), which was confirmed by direct ITC measurements (DeltaC(degree)(p) = +1.941 +/- 0.059 kJ.mol(-1).K(-1)). This unique result appears to come from an extensive additional hydration upon binding and charge-related interactions within the binding site. As a consequence of the positive DeltaC(degree)(p), the nature of the thermodynamic driving force changes with increasing temperature, from enthalpy-driven and entropy-opposed, through enthalpy- and entropy-driven in the range of biological temperatures, into entropy-driven and enthalpy-opposed. Comparison of the van't Hoff and calorimetric enthalpy values provided proof for the ligand protonation at N(1) upon binding, which is required for tight stabilization of the cap-eIF4E complex. Intramolecular self-stacking of the dinucleotide cap-analogue was analyzed to reveal the influence of this coupled process on the thermodynamic parameters of the eIF4E-mRNA 5' cap interaction. The temperature-dependent change in the conformation of 7-methylGpppG shifts significantly the intrinsic DeltaH(degree)(0) = -72.9 +/- 4.2 kJ.mol(-1) and DeltaS(degree)(0) = -116 +/- 58 J.mol(-1).K(-1) of binding to the less negative resultant values, by DeltaH(degree)(sst) = +9.76 +/- 1.15 kJ.mol(-1) and DeltaS(degree)(sst) = +24.8 +/- 2.1 J.mol(-1).K(-1) (at 293 K), while the corresponding DeltaC(degree)(p)(sst) = -0.0743 +/- 0.0083 kJ.mol(-1).K(-1) is negligible in comparison with the total DeltaC(degree)(p) .     

Thermodynamics of the conversion of aqueous xylose to xylulose

The thermodynamics of the conversion of aqueous xylose to xylulose has been investigated using high-pressure liquid chromatography (HPLC) and microcalorimetry. The reaction was carried out in aqueous phosphate buffer over the pH range 6.8-7.4 using solubilized glucose isomerase with MgSO(4) as a cofactor. The temperature range over which this reaction was investigated was 298.15-342.15 K. A combined analysis of both the HPLC and microcalorimetric data leads to the following results at 298.15 K for the conversion process: DeltaG degrees = 4389 +/- 31 J mol(-1), DeltaH degrees = 16090 +/- 670 J mol(-1), and DeltaC(p) degrees = 40 +/- 23 J mol(-1) K(-1). The temperature dependence of the equilibrium constant for the reaction is expressed as R ln K = -4389/298.15 +16090[(1/298.15)-(1/T)]+40[(298.15/T)-1 + ln(T/298.15)]. Comparisons are made with literature data.     

Direct calculation and computer simulation of the enantiomerization barrier of oxazepam in dynamic HPLC experiments--a comparative study

Dynamic chromatographic methods constitute a versatile approach to the rapid and precise determination of enantiomerization barriers of stereolabile drugs. In the present study enantioselective dynamic high-performance liquid chromatography (DHPLC) was employed to determine the enantiomerization barrier of oxazepam. Dynamic elution profiles, exhibiting plateau formation and/or peak broadening between 20 and 60 degrees C at pH 2.6 and pH 8 were obtained in the presence of the chiral stationary phase (CSP) Nucleodex-beta-PM (permethylated beta-cyclodextrin chemically bonded to silica) using a 6:4 mixture of phosphate buffer and methanol as mobile phase. Evaluation of the experimental chromatograms was performed by the novel approximation function (AF) (without computer simulation), and by the stochastic model implemented in the ChromWin simulation software (with computer simulation) furnishing the respective apparent forward rate constants, k(1)(app)(T). From the rate constants, k(1)(app)(T), measured at variable temperatures, the kinetic Eyring activation parameters, deltaG(T)(#), deltaH(#) and deltaS(#), of the enantiomerization of oxazepam were obtained. By variation of the flow rate of the mobile phase, the expected independence of the enantiomerization barrier from the chromatographic time scale was demonstrated for the first time.     

Cytochrome c peroxidase-cytochrome c complex: locating the second binding domain on cytochrome c peroxidase with site-directed mutagenesis

Cytochrome c peroxidase (CcP) can bind as many as two cytochrome c (Cc) molecules in an electrostatic complex. The location of the two binding domains on CcP has been probed by photoinduced interprotein electron transfer (ET) between zinc-substituted horse cytochrome c (ZnCc) and CcP with surface charge-reversal mutations and by isothermal titration calorimetry (ITC). These results, which are the first experimental evidence for the location of domain 2, indicate that the weak-binding domain includes residues 146-150 on CcP. CcP(E290K) has a charge-reversal mutation in the tight-binding domain, which should weaken binding, and it weakens the 1:1 complex; K(1) decreases 20-fold at 18 mM ionic strength. We have employed two mutations to probe the proposed location for the weak-binding domain on the CcP surface: (i) D148K, a "detrimental" mutation with a net (+2) change in the charge of CcP, and (ii) K149E, a "beneficial" mutation with a net (-2) change in the charge. The interactions between FeCc and CcP(WT and K149E) also have been studied with ITC. The CcP(D148K) mutation causes no substantial change in the 2:1 binding but an increase in the reactivity of the 2:1 complex. The latter can be interpreted as a long-range influence on the heme environment or, more likely, the enhancement of a minority subset of binding conformations with favorable pathways for ET. CcP(K149E) has a charge-reversal mutation in the weak-binding domain that produces a substantial increase in the 2:1 binding constant as measured by both quenching and ITC. For the 1:1 complex of CcP(WT), DeltaG(1) = -8.2 kcal/mol (K(1) = 1.3 x 10(6) M(-)(1)), DeltaH(1) = +2.7 kcal/mol, and DeltaS(1) = +37 cal/K.mol at 293 K; for the second binding stage, K(2) < 5 x 10(3) M(-)(1), but accurate thermodynamic parameters were not obtained. For the 1:1 complex of CcP(K149E), DeltaG(1) = -8.5 kcal/mol (K(1) = 2 x 10(6) M(-)(1)), DeltaH(1) = +2. 0 kcal/mol, and DeltaS(1) = +36 cal/K.mol; for the second stage, DeltaG(2) = -5.5 kcal/mol (K(1) = 1.3 x 10(4) M(-)(1)), DeltaH(2) = +2.9 kcal/mol, and DeltaS(2) = +29 cal/K.mol.     

Kinetic and structural characterization of manganese(II)-loaded methionyl aminopeptidases

Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated. Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 6 +/- 0.5 and 1 +/- 0.5 microM for EcMetAP-I and PfMetAP-II, respectively. These K(d) values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 +/- 0.2 and 1.4 +/- 0.2 microM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25-85 degrees C. The activation energy for the Mn(II)-loaded PfMetAP-II hydrolysis of MGMM was found to be 25.7 kJ/mol while the remaining thermodynamic parameters calculated at 25 degrees C are DeltaG+ = 50.1 kJ/mol, DeltaH+ = 23.2 kJ/mol, and DeltaS++ = -90.2 J x mol(-1) x K(-1).     

Influence of pH, temperature, and urea molar flowrate on Arthrospira platensis fed-batch cultivation: a kinetic and thermodynamic approach

Arthrospira platensis was cultivated photoautotrophically at 6.0 klux light intensity in 5.0-L open tanks, using a mineral medium containing urea as nitrogen source. Fed-batch experiments were performed at constant flowrate. A central composite factorial design combined to response surface methodology (RSM) was utilized to determine the relationship between the selected response variables (cell concentration after 10 days, X(m), cell productivity, P(X), and nitrogen-to-cell conversion factor, Y(X/N)) and codified values of the independent variables (pH, temperature, T, and urea flowrate, K). By applying the quadratic regression analysis, the equations describing the behaviors of these responses as simultaneous functions of the selected independent variables were determined, and the conditions for X(m) and P(X) optimization were estimated (pH 9.5, T = 29 degrees C, and K = 0.551 mM/day). The experimental data obtained under these conditions (X(m) = 749 mg/L; P(X) = 69.9 mg/L.day) were very close to the estimated ones (X(m) = 721 mg/L; P(X) = 67.1 mg/L.day). Additional cultivations were carried out under the above best conditions of pH control and urea flowrate at variable temperature. Consistently with the results of RSM, the best growth temperature was 29 degrees C. The maximum specific growth rates at different temperatures were used to estimate the thermodynamic parameters of growth (DeltaH* = 59.3 kJ/mol; DeltaS* = -0.147 kJ/mol.K; DeltaG* = 103 kJ/mol) and its thermal inactivation (DeltaH(D) (o) = 72.0 kJ/mol; DeltaS(D) (o) = 0.144 kJ/mol.K; DeltaG(D) (o) = 29.1 kJ/mol).     

Detergent-like action of the antibiotic peptide surfactin on lipid membranes

Surfactin is a bacterial lipopeptide with powerful surfactant-like properties. High-sensitivity isothermal titration calorimetry was used to study the self association and membrane partitioning of surfactin. The critical micellar concentration (CMC), was 7.5 microM, the heat of micellization was endothermic with DeltaH(w-->m)(Su) = +4.0 kcal/mol, and the free energy of micellization DeltaG(O,w-->m)(Su) = -9.3 kcal/mol (25 degrees C; 100 mM NaCl; 10 mM TRIS, 1 mM EDTA; pH 8.5). The specific heat capacity of micellization was deduced from temperature dependence of DeltaH(w-->m)(Su) as DeltaC(w-->m)(P) = -250 +/- 10 cal/(mol.K). The data can be explained by combining the hydrophobicity of the fatty acyl chain with that of the hydrophobic amino acids. The membrane partition equilibrium was studied using small (30 nm) and large (100 nm) unilamellar POPC vesicles. At 25 degrees C, the partition coefficient, K, was (2.2 +/- 0.2) x 10(4) M(-1) for large vesicles leading to a free energy of DeltaG(O, w-->b)(Su) = -8.3 kcal/mol. The partition enthalpy was again endothermic, with DeltaH(w-->b)(Su) = 9 +/- 1 kcal/mol. The strong preference of surfactin for micelle formation over membrane insertion explains the high membrane-destabilizing activity of the peptide. For surfactin and a variety of non-ionic detergents, the surfactant-to-lipid ratio, inducing membrane solubilization, R(sat)(b), can be predicted by the simple relationship R(sat)(b) approximately K. CMC.     

Overexpression and divalent metal binding properties of the methionyl aminopeptidase from Pyrococcus furiosus

The gene encoding for the methionyl aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II; EC 3.4.11.18) has been inserted into a pET 27b(+) vector and overexpressed in Escherichia coli. The new expression system resulted in a 5-fold increase in purified enzyme obtained from a 5 L fermentor growth. The as-purified PfMetAP-II enzyme, to which no exogenous metal ions or EDTA was added, was found to have 1.2 equiv of zinc and 0.1 equiv of iron present by ICP-AES analysis. This enzyme had a specific activity of 5 units/mg, a 60-fold decrease from the fully loaded Fe(II) enzymes. When an additional 2 equiv of Zn(II) was added to the as-purified PfMetAP-II, no activity could be detected. The combination of these data with previously reported whole cell studies on EcMetAP-I further supports the suggestion that the in vivo metal ion for all MetAP's is Fe(II). Both Co(II)- and Fe(II)-loaded PfMetAP-II showed similar substrate specificities to EcMetAP-I. Substrate binding was largely affected by the amino acid in the P1 position and the length of the polypeptide. The substrates MSSHRWDW and MP-p-NA showed the smallest K(m) values while the substrates MGMM and MP-p-NA provided the highest turnover. The catalytic efficiency (k(cat)/K(m)) of PfMetAP-II for MP-p-NA at 30 degrees C was 799 500 and 340 930 M(-1) s(-1) for Co(II)- and Fe(II)-loaded PfMetAP-II, respectively. Maximum catalytic activity was obtained with 1 equiv of Co(II) or Fe(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 50 +/- 15 and 20 +/- 15 nM for Co(II)- and Fe(II)-substituted PfMetAP-II, respectively. Electronic absorption spectral titration of a 1 mM sample of apo-PfMetAP-II with Co(II) provided a dissociation constant of 0.35 +/- 0.02 mM for the second metal binding site, a 17500-fold increase compared to the first metal binding site. The electronic absorption data also indicated that both Co(II) ions reside in a pentacoordinate geometry. PfMetAP-II shows unique thermostability and the optimal temperature for substrate turnover was found to be approximately 85 degrees C at pH 7.5 in 25 mM Hepes and 150 mM KCl buffer. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the k(cat) values and was found to be linear over the temperature range 25-85 degrees C, indicating that the rate-limiting step in PfMetAP-II peptide hydrolysis does not change as a function of temperature. Co(II)- and Fe(II)-loaded PfMetAP-II have similar activation energies (13.3 and 19.4 kJ/mol, respectively). The thermodynamic parameters calculated at 25 degrees C are as follows: DeltaG++ = 46.23 kJ/mol, DeltaH++ = 10.79 kJ/mol, and DeltaS++ = -119.72 J.mol(-1).K(-1) for Co(II)-loaded PfMetAP; DeltaG++ = 46.44 kJ/mol, DeltaH++ = 16.94 kJ/mol, and DeltaS++ = -99.67 J.mol(-1).K(-1) for Fe(II)-loaded PfMetAP. Interestingly, at higher temperatures (> 50 degrees C), Fe(II)-loaded PfMetAP-II is more active (1.4-fold at 85 degrees C) than Co(II)-loaded PfMetAP-II.     

Thermodynamics of the arginine kinase reaction.

The effect of temperature, pH, free [Mg(2+)], and ionic strength on the apparent equilibrium constant of arginine kinase (EC 2.7.3.3) was determined. At equilibrium, the apparent K' was defined as [see text] where each reactant represents the sum of all the ionic and metal complex species. The K' at pH 7.0, 1.0 mM free [Mg(2+)], and 0. 25 M ionic strength was 29.91 +/- 0.59, 33.44 +/- 0.46, 35.44 +/- 0. 71, 39.64 +/- 0.74, and 45.19 +/- 0.65 (n = 8) at 40, 33, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy (DeltaH degrees') is -8.19 kJ mol(-1), and the corresponding standard apparent entropy of the reaction (DeltaS degrees') is + 2. 2 J K(-1)mol(-1) in the direction of ATP formation at pH 7.0, free [Mg(2+)] =1.0 mM, ionic strength (I) =0.25 M at 25 degrees C. We further show that the magnitude of transformed Gibbs energy (DeltaG degrees ') of -8.89 kJ mol(-1) is mostly comprised of the enthalpy of the reaction, with 7.4% coming from the entropy TDeltaS degrees' term (+0.66 kJ mol(-1)). Our results are discussed in relation to the thermodynamic properties of its evolutionary successor, creatine kinase.     

Characterization of the catalytically active Mn(II)-loaded argE -encoded N -acetyl-l -ornithine deacetylase from Escherichia coli

The catalytically competent Mn(II)-loaded form of the argE-encoded N-acetyl-l-ornithine deacetylase from Escherichia coli (ArgE) was characterized by kinetic, thermodynamic, and spectroscopic methods. Maximum N-acetyl-l-ornithine (NAO) hydrolytic activity was observed in the presence of one Mn(II) ion with k _cat and K _m values of 550 s⁻¹ and 0.8 mM, respectively, providing a catalytic efficiency (k _cat/K _m) of 6.9 × 10⁵ M⁻¹ s⁻¹. The ArgE dissociation constant (K _d) for Mn(II) was determined to be 0.18 μM, correlating well with a value obtained by isothermal titration calorimetry of 0.30 μM for the first metal binding event and 5.3 μM for the second. An Arrhenius plot of the NAO hydrolysis for Mn(II)-loaded ArgE was linear from 15 to 55 °C, suggesting the rate-limiting step does not change as a function of temperature over this range. The activation energy, determined from the slope of this plot, was 50.3 kJ mol⁻¹. Other thermodynamic parameters were ΔG ^‡ = 58.1 kJ mol⁻¹, ΔH ^‡ = 47.7 kJ mol⁻¹, and ΔS ^‡ = –34.5 J mol⁻¹ K⁻¹. Similarly, plots of lnK _m versus 1/T were linear, suggesting substrate binding is controlled by a single step. The natural product, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]leucine (bestatin), was found to be a competitive inhibitor of ArgE with a K _i value of 67 μM. Electron paramagnetic resonance (EPR) data recorded for both [Mn(II)_(ArgE)] and [Mn(II)Mn(II)(ArgE)] indicate that the two Mn(II) ions form a dinuclear site. Moreover, the EPR spectrum of [Mn(II)Mn(II)(ArgE)] in the presence of bestatin indicates that bestatin binds to ArgE but does not form a μ-alkoxide bridge between the two metal ions.     

Thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia

The thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia have been investigated using both heat conduction microcalorimetry and high-pressure liquid chromatography. The reaction was carried out in aqueous phosphate buffer over the pH range 7.25-7.43, the temperature range 13-43 degrees C, and at ionic strengths varying from 0.066 to 0.366 mol kg(-1). The following values have been found for the conversion of aqueous L-aspartateH- to fumarate2- and NH4+ at 25 degrees C and at zero ionic strength: K = (1.48 +/- 0.10) x 10(-3), DeltaG degrees = 16.15 +/- 0.16 kJ mol(-1), DeltaH degrees = 24.5 +/- 1.0 kJ mol(-1), and DeltaC(p) degrees = -147 +/- 100 J mol(-1) K(-1). Calculations have also been performed which give values of the apparent equilibrium constant for the conversion of L-aspartic acid to fumaric acid and ammonia as a function of temperature, pH and ionic strength.     

Kinetic and thermodynamic studies of tet repressor-tetracycline interaction

Stopped-flow measurements have been employed to study the kinetics of the conformational changes in TetR (B) induced by tetracycline binding with and without Mg(2+) ions. Result of stopped-flow fluorometry measurements at pH 8.0 indicate conformational changes in the helix-turn-helix motif in the N-terminal domain and in the C-terminal inducer binding domain. Binding of tetracycline (Tc) to TetR in the absence of Mg(2+) can be described by a simple kinetics process, which is limited to the first step association without any unimolecular conformational change step upon Tc binding. The rate constants for this process are equal to 2.0 x 10(5) M(-)(1) s(-)(1) and 2.1 s(-)(1) for the forward and backward reaction, respectively, and gave the binding constant K(a) = 0.96 x 10(5) M(-)(1). The kinetics of [Tc-Mg](+) binding to TetR can be described by reactions in which the first step describes the association characterized by the rate constants k(a) = 1.4 x 10(5) M(-)(1) s(-)(1) and k(d) = 2.2 x 10(-)(2) s(-)(1) and binding constant K(a) = 6.3 x 10(6) M(-)(1). The first step of [Tc-Mg](+) association is followed by at least three conformational change steps, which occur in the inducer binding site and then propagate to the surroundings of Trp75 and Trp43 residues. The rate constants for the forward, k(c), and backward, k(-)(c), reaction for each of these conformational steps have been determined. The thermodynamics of the binding of tetracycline with and without Mg(2+) to TetR was investigated by isothermal titration calorimetry (ITC) at pH 8.0 and 25 degrees C. The measurement shows that TetR dimer possesses two equivalent binding sites for tetracycline, characterized by binding constant K(a) = 9.0 x 10(6) M(-)(1) and K(a) = 7.0 x 10(4) M(-)(1) for Tc with and without Mg(2+), respectively. The binding of the inducer to TetR, in the presence and absence of Mg(2+) ion, is an enthalpy-driven reaction characterized by DeltaH = -51 kJ mol(-)(1) and DeltaH = -33 kJ mol(-)(1), respectively. The entropy change, DeltaS, for the interaction in the presence of Mg(2+) is equal to -38.9 J K(-)(1) mol(-)(1), and for the tetracycline alone, it was estimated at -17.6 J K(-)(1) mol(-)(1).     

Multi-method global analysis of thermodynamics and kinetics in reconstitution of monellin

Accurate and precise determinations of thermodynamic parameters of binding are important steps toward understanding many biological mechanisms. Here, a multi-method approach to binding analysis is applied and a detailed error analysis is introduced. Using this approach, the binding thermodynamics and kinetics of the reconstitution of the protein monellin have been quantitatively determined in detail by simultaneous analysis of data collected with fluorescence spectroscopy, surface plasmon resonance and isothermal titration calorimetry at 25 degrees C, pH 7.0 and 150 mM NaCl. Monellin is an intensely sweet protein composed of two peptide chains that form a single globular domain. The kinetics of the reconstitution reaction are slow, with an association rate constant, k(on) of 8.8 x 10(3) M(-1) s(-1) and a dissociation rate constant, k(off) of 3.1 x 10(-4) s(-1). The equilibrium constant K(A) is 2.8 x 10(7) M(-1) corresponding to a standard free energy of association, DeltaG degrees , of -42.5 kJ/mol. The enthalpic component, DeltaH degrees , is -18.7 kJ/mol and the entropic contribution, DeltaS degrees , is 79.8 J mol(-1) K(-1) (-TDeltaS degrees = -23.8 kJ/mol). The association of monellin is therefore a bimolecular intra-protein association whose energetics are slightly dominated by entropic factors.     

Thermodynamics of CFTR channel gating: a spreading conformational change initiates an irreversible gating cycle

CFTR is the only ABC (ATP-binding cassette) ATPase known to be an ion channel. Studies of CFTR channel function, feasible with single-molecule resolution, therefore provide a unique glimpse of ABC transporter mechanism. CFTR channel opening and closing (after regulatory-domain phosphorylation) follows an irreversible cycle, driven by ATP binding/hydrolysis at the nucleotide-binding domains (NBD1, NBD2). Recent work suggests that formation of an NBD1/NBD2 dimer drives channel opening, and disruption of the dimer after ATP hydrolysis drives closure, but how NBD events are translated into gate movements is unclear. To elucidate conformational properties of channels on their way to opening or closing, we performed non-equilibrium thermodynamic analysis. Human CFTR channel currents were recorded at temperatures from 15 to 35 degrees C in inside-out patches excised from Xenopus oocytes. Activation enthalpies(DeltaH(double dagger)) were determined from Eyring plots. DeltaH(double dagger) was 117 +/- 6 and 69 +/- 4 kJ/mol, respectively, for opening and closure of partially phosphorylated, and 96 +/- 6 and 73 +/- 5 kJ/mol for opening and closure of highly phosphorylated wild-type (WT) channels. DeltaH(double dagger) for reversal of the channel opening step, estimated from closure of ATP hydrolysis-deficient NBD2 mutant K1250R and K1250A channels, and from unlocking of WT channels locked open with ATP+AMPPNP, was 43 +/- 2, 39 +/- 4, and 37 +/- 6 kJ/mol, respectively. Calculated upper estimates of activation free energies yielded minimum estimates of activation entropies (DeltaS(double dagger)), allowing reconstruction of the thermodynamic profile of gating, which was qualitatively similar for partially and highly phosphorylated CFTR. DeltaS(double dagger) appears large for opening but small for normal closure. The large DeltaH(double dagger) and DeltaS(double dagger) (TDeltaS(double dagger) >/= 41 kJ/mol) for opening suggest that the transition state is a strained channel molecule in which the NBDs have already dimerized, while the pore is still closed. The small DeltaS(double dagger) for normal closure is appropriate for cleavage of a single bond (ATP's beta-gamma phosphate bond), and suggests that this transition state does not require large-scale protein motion and hence precedes rehydration (disruption) of the dimer interface.     

Measurement of thermodynamic parameters for hydrophobic mismatch 1: self-association of a transmembrane helix

Membrane partitioning and self-association of transmembrane helices are crucial thermodynamic steps for membrane protein folding, although experimental difficulties have hampered quantitative estimations of related thermodynamic parameters, especially in lipid bilayer environments. This article reports for the first time, the complete set of thermodynamic parameters (DeltaG, DeltaH, DeltaS, and DeltaC(p)) for the formation of the antiparallel dimer of the inert hydrophobic model transmembrane helix X-(AALALAA)(3)-Y (X = 7-nitro-2-1, 3-benzoxadiazol-4-yl (NBD) and Y = NH(2) (I) or X = Ac and Y = NHCH(2)CH(2)-S-N-[4-[[4-(dimethylamino)phenyl]azo]phenyl]maleimide (DABMI) (II)) in dimonounsaturated phosphocholine lipid bilayers with different hydrophobic thicknesses (C14-C22) at 5-55 degrees C, as evaluated by fluorescence resonance energy transfer from I to II. Stronger dimerization was observed in thicker membranes and at lower temperatures (DeltaG = -9 to -26 kJ mol(-)(1)), driven by large negative DeltaH values (-18 to -80 kJ mol(-)(1)). Fourier transform infrared-polarized spectroscopy revealed that the peptide formed a stable transmembrane helix with an orientation angle of approximately 15 degrees in all bilayers without significant effects on lipid structures, suggesting that the depth to which the helix termini penetrate changes depending on the degree of hydrophobic mismatch. The enthalpy changes for helix-helix interactions can be well explained by the electrostatic interactions between helix macrodipoles in different dielectric environments. The new concept of dipole-dipole interaction as a basic driving force of helix dimerization will become a basis for understanding the structural and functional modifications in response to hydrophobic mismatch.