Enzymatic synthesis of β-lactam antibiotics: A thermodynamic background

The equilibrium constants and the respective standard Gibbs energy changes for hydrolysis of some β-lactam antibiotics have been determined. Native and immobilized penicillin amidase (EC 3.5.1.11) from Escherichia coli has been used as a catalyst. The values of standard Gibbs energy changes corresponding to the pH-independent product of equilibrium concentrations (ΔG⁰_c = ? RT ln K_c) have been calculated. The differences in the structure of the antibiotics nucleus hardly ever affect the value of the pH-independent component of the standard Gibbs energy change (ΔG⁰_c) and value of apparent standard Gibbs energy change at a fixed pH (ΔG^0′_c). At the same time, the value of ΔG⁰_c is more sensitive to the structure of the acyl moiety of the antibiotic; when ampicillin is used instead of benzylpenicillin, ΔG⁰_c increases by ～6.3 kJ mol^?1 (1.5 kcal mol^?1). pH-dependences of the apparent standard Gibbs energy changes for hydrolysis of β-lactam antibiotics have been calculated. The pH-dependences of ΔG^0′_c for hydrolysis of all β-lactam antibiotics have a similar pattern. The thermodynamic pH optimum of the synthesis of these compounds is in the acid pH range (pH < 5.0). The breakage of the β-lactam ring leads to a sharp decrease in the ΔG^0′_c value and a change in the pattern of the pH-dependence. For example, at pH 5.0 ΔG^0′_c decreases from 14.4 kJ mol^?1 for benzylpenicillin to ?1.45 kJ mol^?1 for benzylpenicilloic acid. The reason for these changes is mainly a considerable increase in the pK of the amino group of the nucleus of the antibiotic and, as a consequence, a decrease in the component of standard Gibbs energy change, corresponding to the ionization of the system. The thermodynamic potentials of the enzymatic synthesis of semisynthetic penicillins and cephalosporins on the basis of both free acids and their derivatives (N-acylated amino acids, esters) are discussed. It is shown that with esters of the acids, a high yield of the antibiotic can, in principle, be achieved at higher pH values.     

Calorimetric studies of oxyhemoglobin dissociation. I. Reaction of sodium dithionite with oxygen

Calorimetric studies of the reduction of free oxygen in solution by sodium dithionite are in agreement with a stoichiometry of 2 moles Na₂S₂O₄ per mole of oxygen. The reaction is biphasic with ΔH_t - 118±7 kcal mol^?1 (?494 ± 29 kJ mol^?1). The initial phase of the reaction proceeds with an enthalpy change of ca ?20 kcal (?84 kJ) and occurs when 0.5 moles of dithionite have been added per mole dioxygen present. This could be interpreted as the enthalpy change for the addition of a single electron to form the superoxide anion. Further reduction of the oxygen to water by one or more additional steps is accompanied by an enthalpy change of ca ?100 kcal (?418. 5 kJ). Neither of these reductive phases is consistent with the formation of hydrogen peroxide as an intermediate. The reduction of hydrogen peroxide by dithionite in 0.1 M phosphate buffer, pH 7.15, is a much slower process and with an enthalpy change of ca ? 74 kcal mol^?1 (?314 kJ mol^?1). Dissociation of oxyhemoglobin induced by the reduction of free oxygen tension with dithionite also shows a stoichiometry of 2 moles dithionite per mole oxygen present and an enthalpy change of ca. ?101 ±9 kcal mol^?1 (?423± 38 kJ mol^?1). The difference in the observed enthalpies (reduction of dioxygen vs. oxyhemoglobin) has been attributed to the dissociation of oxyhemoglobin, which is 17 kcal mol^?1 (71 kJ mol^?1).     

A calorimetric study of the binding of 2'-deoxyuridine-5'-phosphate and 5-fluoro-2'-deoxyuridine-5'-phosphate to thymidylate synthetase.

The thermodynamic parameters, ΔH′, ΔG′, and ΔS′, and the stoichiometry for the binding of the substrate 2′-deoxyuridine-5′-phosphate (dUMP) and the inhibitor 5-fluoro-2′-deoxyuridine-5′-phosphate (FdUMP) to Lactobacillus casei thymidylate synthetase (TSase) have been investigated using both direct calorimetric methods and gel filtration methods. The data obtained show that two ligand binding sites are available but that the binding of the second mole of dUMP is extremely weak. Binding of the first mole of dUMP can best be illustrated by dUMP + TSase + H⁺?(dUMP-TSase-H⁺). [1] The enthalpy, ΔH₁′, for reaction [1] was measured directly on a flow modification of a Beckman Model 190B microcalorimeter. Experiments in two different buffers (I = 0.10 m) show that ΔH₁′ = ?28 kJ mol^?1 and that 0.87 mol of protons enters into the reaction. Analysis of thermal titrations for reaction [1] indicates a free energy change of ΔG₁′ = ?30 kJ mol^?1 (K₁ = 1.7 × 10⁵ m^?1). From these parameters, ΔS₁′ was calculated to be +5 J mol^?1 degree^?1, showing that the reaction is almost totally driven by enthalpy changes. Gel filtration experiments show that at very high substrate concentrations, binding to a second site can be observed. Gel filtration experiments performed at low ionic strength (I = 0.05 m) reveal a stronger binding, with ΔG₁′ = ?35 kJ mol^?1 (K₁ = 1.2 × 10⁶ m^?1), suggesting that the forces driving the interaction are, in part, electrostatic. Addition of 2-mercaptoethanol (0.10 m) had the effect of slightly increasing the dUMP binding constant. Binding of FdUMP to TSase is best illustrated by 2FdUMP + TSase + n_HH⁺?FdUMP₂ ? TSase ? (H⁺)_nH. [2] The enthalpy for this reaction, ΔH₂, was also measured calorimetrically and found to be ?30 kJ mol^?1 with n_H = 1.24 at pH 7.4 Assuming two FdUMP binding sites per dimer as established by Galivan et al. [Biochemistry15, 356–362 (1976)] our calorimetric results indicate different binding energies for each site. Based on the binding data, a thermodynamic model is presented which serves to rationalize much of the confusing physical and chemical data characterizing thymidylate synthetase.     

DFT and docking studies of rhodostreptomycins A and B and their interactions with solvated/nonsolvated Mg2+ and Ca2+ ions

The interactions of L-aminoglucosidic stereoisomers such as rhodostreptomycins A (Rho A) and B (Rho B) with cations (Mg²⁺, Ca²⁺, and H⁺) were studied by a quantum mechanical method that utilized DFT with B3LYP/6-311G**. Docking studies were also carried out in order to explore the surface recognition properties of L-aminoglucoside with respect to Mg²⁺ and Ca²⁺ ions under solvated and nonsolvated conditions. Although both of the stereoisomers possess similar physicochemical/antibiotic properties against Helicobacter pylori, the thermochemical values for these complexes showed that its high affinity for Mg²⁺ cations caused the hydration of Rho B. According to the results of the calculations, for Rho A–Ca²⁺(H₂O)₆, ΔH = ?72.21 kcal?mol^?1; for Rho B–Ca²⁺(H₂O)₆, ΔH = ?72.53 kcal?mol^?1; for Rho A–Mg²⁺(H₂O)₆, ΔH = ?72.99  kcal?mol^?1 and for Rho B–Mg²⁺(H₂O)₆, ΔH = ?95.00  kcal?mol^?1, confirming that Rho B binds most strongly with hydrated Mg²⁺, considering the energy associated with this binding process. This result suggests that Rho B forms a more stable complex than its isomer does with magnesium ion. Docking results show that both of these rhodostreptomycin molecules bind to solvated Ca²⁺ or Mg²⁺ through hydrogen bonding. Finally, Rho B is more stable than Rho A when protonation occurs.

Purification,kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.)

We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: k_m = 55 mg ml^?1, k_cat = 21 s^?1, k_cat/k_m = 0.38, while the thermodynamic parameters were: ΔH* = 52.6 kJ mol^?1, ΔG* = 71.2 kJ mol^?1, ΔS* = ?57 J mol^?1 K^?1, ΔG*_E–S = 10.8 kJ mol^?1 and ΔG*_E–T = 2.6 kJ mol^?1. The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53–63 °C were also determined. The half -life of SAI at 53 and 63 °C was 112 and 10 min, respectively. At 55 °C, surprisingly the half -life increased to twice that at 53 °C. ΔG*, ΔH* and ΔS* of irreversible thermal stability of SAI at 55 °C were 107.7 kJ mol^?1, 276.04 kJ mol^?1 and 513 J mol^?1K^?1, respectively.     

Thermodynamics of base interaction in (A)n and (A.U)n

Using precision scanning microcalorimetry we studied (A)_n and (A·U)_n melting in highly diluted solutions (0.3 to 5.0 mm) with different Na⁺ activity. This permitted us to determine directly the thermodynamic functions of stacking interaction in (A)_n and base-pairing in (A·U)_n. For (A-A) stacking at (A)_n melting temperature we obtained ΔH^(A)n_m = 12.6 kJ mol^?1; ΔS^(A)n_m = 41 J K^?1 mol^?1. For A·U base-pairing at a standard temperature of 298 K and 0.1 m-Na⁺ we have: ΔH^(A·U) = 34 kJ mol^?1; ΔS^(A·U) = 102 J K^?1 mol^?1ΔG^(A·U) = ?3.5 kJ mol^?1.     

The enthalpy of oxidation of flavin mononucleotide: Temperature dependence of in vitro bacterial luciferase bioluminescence

The enthalpy of the bioluminescent reaction

$\text{FMNH}{}_2\text{+ RCHO + O}{}_2\text{→}\text{luciferase}\text{FMN + RCOO + H}{}_3\text{O}{}^{\mathrm{+}}\text{+ hv}$

has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH₂) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔH_L = ?338.9 k.J (mol FMN)^?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)^?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔG_L = ?464.8 kJ (mol FMN)^?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH₂ + O₂ ? FMN + H₂O₂; ΔH_D = ?158.5 kJ (mol FMN)^?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG₁, and intrinsic enthalpy, ΔH₁, which are functionally defined by the relations ΔG_I = ΔG_L ? uhvΔH_I = ΔH_L ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole^?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)^?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission.     

Retention of calcium by mitochondria isolated from Ehrlich ascites tumor cells

Tightly coupled mitochondria isolated from Ehrlich ascites tumor cells accumulate and retain high concentrations of Ca²⁺ in the presence of ATP for periods up to at least 20 min at 25 °C. The presence of inorganic phosphate up to 20 mm does not prevent such Ca²⁺ retention. The tumor mitochondria accumulate Ca²⁺ in the presence of succinate as an energy source but lose the Ca²⁺ after 1–2 min. Addition of ATP (K_m approx 1 mm) to the incubation medium after Ca²⁺ release, induces reaccumulation of the ion. Thus, the ability of the tumor mitochondria to retain Ca²⁺ differs markedly from that of rat liver mitochondria and is seen as being of potential biological significance to the unique metabolic behavior of the ascites tumor cells.     

Conformational stability of α-amylase from malted sorghum (Sorghum bicolor): Reversible unfolding by denaturants

α-Amylase from Sorghum bicolor, is reversibly unfolded by chemical denaturants at pH 7.0 in 50 mM Hepes containing 13.6 mM calcium and 15 mM DTT. The isothermal equilibrium unfolding at 27 °C is characterized by two state transition with ΔG (H₂O) of 16.5 kJ mol⁻¹ and 22 kJ mol⁻¹, respectively, at pH 4.8 and pH 7.0 for GuHCl and ΔG (H₂O) of 25.2 kJ mol⁻¹ at pH 4.8 for urea. The conformational stability indicators such as the change in excess heat capacity (ΔC_p), the unfolding enthalpy (H_g) and the temperature at ΔG = 0 (T_g) are 17.9 ± 0.7 kJ mol⁻¹ K⁻¹, 501.2 ± 18.2 kJ mol⁻¹ and 337.3 ± 6.9 K at pH 4.8 and 14.3 ± 0.5 kJ mol⁻¹ K⁻¹, 509.3 ± 21.7 kJ mol⁻¹ and 345.4 ± 4.8 K at pH 7.0, respectively. The reactivity of the conserved cysteine residues, during unfolding, indicates that unfolding starts from the ‘B’ domain of the enzyme. The oxidation of cysteine residues, during unfolding, can be prevented by the addition of DTT. The conserved cysteine residues are essential for enzyme activity but not for the secondary and tertiary fold acquired during refolding of the denatured enzyme. The pH dependent stability described by ΔG (H₂O) and the effect of salt on urea induced unfolding confirm the role of electrostatic interactions in enzyme stability.     

Proton magnetic resonance study of rotation about carbon-nitrogen bonds in pentakis(1,1-dimethylurea)dioxouranium(VI) and its 1,3-dimethylurea analogue

In CD₃CN solutions the kinetic parameters characterising rotation about the CNMe₂ and CNH₂ bonds in [UO₂(1,1-DMU)₅]²⁺ (1,1-DMU = 1,1- dimethylurea) were determined as: k(265 K) = 39.1 ± 0.4 and 2960 ± 60 s^?1, ΔH3 = 49.1 ± 0.76 and 61.1 ±0.5 kJ mol^?1, ΔS2 = ?28.3 ± 2.7 and 53.1 ± 2.2 J K^?1 mol^?1 respectively from ¹H NMR studies. Resonances arising from the three isomeric 1,3-DMU (= 1,3-dimethylurea) ligands were observed for [UO₂(1,3-DMU)₅]²⁺ in CD₃CN solution and the kinetic parameters characterising their isomerisations were also determined. The three isomers of 1,3-DMU have not previously been detected in solution and it appears that coordination of 1,3-DMU to UO₂²⁺ increases the barrier to rotation about the carbon nitrogen bond, as is also shown to be the case for 1,1-DMU.     

Denaturation thermodynamics of chicken cardiac metmyoglobin

The unfolding at pH 8 of chicken cardiac aquometmyoglobin was examined as a function of temperature and concentration of guanidinium chloride using the two-state model. The isothermal unfolding data at 25°C were fitted to Tanford's transfer model and the binding model of Aune and Tanford. The estimates obtained for ΔG_D) were virtually identical, viz., 8.3 ±0.3 kcal mol^?1. The chicken metmyoglobin is thus some 5.3 kcal mol^?1 less stable than that of sperm whale metmyoglobin. The unfolding parameters α and Δn were decreased 20% from those of mammalian myoglobins thus far examined, suggesting nonidentity of native conformations. The apparent enthalpy change on unfolding was dependent on both temperature and denaturant concentration. The decreases in the isothermal unfolding parameters from those of sperm whale are principally assigned to three of the 46 sequence changes.     

Thermodynamic interaction between urea and the peptide group in aqueous solutions at 25°C

Isopiestic vapor pressure measurements of the ternary systems water + triglycine + urea and water + glycine-L-alanine + urea were made and used to calculate the Gibbs free energy of these systems. Together with recently published analogous results on systems, in which the first solute was glycine or alanine or diglycine, and measurements of the excess enthalpy of all these solutions, it is possible to calculate the Gibbs free energy of transfer and the enthalpy of transfer of the peptide group from water to aqueous urea solutions. The transfer can be described as a binding of urea to the peptide group with ΔG = ?1.85 kJ mol^?1 and ΔH = ?18.7 kJ mol^?1 at 298.1 K.     

Thermodynamics and coordination characteristics of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 extraction complex

The extraction equilibrium of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 complex was carried out in the crown ether1,2-dichloroethaneHCl aqueous solution system at different temperatures. The extraction complex has the overall composition (L)₂·(H₃O⁺·χH₂O)₂·UO₂Cl₄²⁻ (L = dicyclohexano-24-crown-8). The values of the extraction equilibrium constants (K_ex) increase steadily with a decrease in temperature: 13.5 (298 K), 7.96 (301 K), 4.20 (303 K) and 2.07 (305 K). A plot of log K_ex against 1/T shows a straight line. The value of the enthalpy change, ΔH°, was calculated from the slope and equals −212 kJ mol⁻¹. The value of the entropy change, ΔS°, was calculated from ΔH° and K_ex and equals −690 J K⁻¹ mol⁻¹, whereas ΔG° = −6.45 kJ mol⁻¹. Comparing these thermodynamic parameters with those of the dicyclohexano-18-crown-6 isomer A [1] (ΔS° = −314 J K⁻¹ mol⁻¹, ΔH° = −101 kJ mol⁻¹ and ΔG° = −8.37 kJ mol⁻¹), it can be seen that ΔH° and ΔS° are more negative for the former than for the latter, and both are enthalpy-stabilized complexes. The molecular structure of the complex has the feature that there are two H₅O₂⁺ ions in it, in contrast to the H₃O⁺ ions in the dicyclohexano-18-crown-6 isomer A complex [1]. Each of the H₅O₂⁺ ions is held in the crown ether cavity by four hydrogen bonds. The H₅O₂⁺ ion has a central bond. The uranium atom forms UO₂Cl₄²⁻ as a counterion away from the crown ether. The formation of this complex is in good agreement with more negative entropy change and less negative free energy change, as mentioned above.     

Kinetics and thermodynamics of catalysis and thermal inactivation of a novel α-amylase (Tp-AmyS) from Thermotoga petrophila

Abstract

This research is focussed on kinetic, thermodynamic and thermal inactivation of a novel thermostable recombinant α-amylase (Tp-AmyS) from Thermotoga petrophila. The amylase gene was cloned in pHIS-parallel1 expression vector and overexpressed in Escherichia coli. The steady-state kinetic parameters (V_max, K_m, k_cat and k_cat/K_m) for the hydrolysis of amylose (1.39?mg/min, 0.57?mg, 148.6?s^?1, 260.7), amylopectin (2.3?mg/min, 1.09?mg, 247.1?s^?1, 226.7), soluble starch (2.67?mg/min, 2.98?mg, 284.2?s^?1, 95.4) and raw starch (2.1?mg/min, 3.6?mg, 224.7?s^?1, 61.9) were determined. The activation energy (E_a), free energy (ΔG), enthalpy (ΔH) and entropy of activation (ΔS) at 98?°C were 42.9?kJ mol^?1, 74?kJ mol^?1, 39.9?kJ mol^?1 and ?92.3 J mol^?1 K^?1, respectively, for soluble starch hydrolysis. While ΔG of substrate binding (ΔG_E-S) and ΔG of transition state binding (ΔG_E-T) were 3.38 and ?14.1?kJ mol^?1, respectively. Whereas, EaD, Gibbs free energy (ΔG*), increase in the enthalpy (ΔH*) and activation entropy (ΔS*) for activation of the unfolding of transition state were 108, 107, 105?kJ mol^?1 and ?4.1 J mol^?1 K^?1. The thermodynamics of irreversible thermal inactivation of Tp-AmyS revealed that at high temperature the process involves the aggregation of the protein.     

Effect of trifluoperazine on skeletal muscle mitochondrial respiration

The effect of trifluoperazine on the respiration of porcine liver and skeletal muscle mitochondria was investigated by polarographic and spectroscopic techniques. Low concentrations of trifluoperazine (88 nmol/mg protein) inhibited both the ADP- and Ca²⁺-stimulated oxidation of succinate, and reduced the values of the respiratory control index and the $\text{ADP}\text{O}$ and $\text{Ca}{}^{\mathrm{2+}}\text{O}$ ratio. High concentrations inhibited both succinate and ascorbate plus tetramethyl-p-phenylenediame (TMPD) oxidations, and uncoupler (carbonyl cyanide p-trifluromethoxyphenylhydrazone) and Ca²⁺-stimulated respiration. Porcine liver mitochondria were more sensitive to trifluoperazine than skeletal muscle mitochondria. Trifluoperazine inhibited the electron transport of succinate oxidation of skeletal muscle mitochondria within the cytochrome b-c₁ and cytochrome c₁-aa₃ segments of the respiratory chain system. 233 nmol trifluoperazine/mg protein inhibited the aerobic steady-state reduction of cytochrome c₁ by 92% with succinate as substrate, and of cytochrome c and cytochrome aa₃ by 50–60% with ascorbate plus TMPD as electron donors. Trifluoperazine can thus inhibit calmodulin-independent reactions particularly when used at high concentrations.     

Kinetics of selenite uptake by mononuclear cells from peripheral human blood

The present study deals with the kinetics and thermodynamics of the uptake of⁷⁵Se-labeled SeO ₃ ^2? from incubation media to lymphocytes cultivated from eight normal individuals (14–55 years of age, two females). The uptake of SeO ₃ ^2? was evaluated on the assumption of pseudo-first-order kinetics with regard to a reacting cellular receptor pool. On the basis of the experimental observations, it was assumed that the suggested pool of receptor molecules-symbolically represented by “£H₄”—reacts with SeO ₃ ^2? in the hypothetical reaction: $$\pounds H_4 + SeO_3^{2 - } + 2H^ + \underset{{ - k_1 }}{\overset{{k_1 }}{\longleftrightarrow}}\pounds Se + 3H_2 O$$ The mean value of the change in standard free energy at 25°C was calculated to be ΔG ^o=?141.6±1.3 kJ/mol, while the corresponding mean value of the free energy of activation at 25°C was calculated to be ΔG ²⁺=?7.8±0.9 kJ/mol for the forward reaction. The calculated values of the corresponding individual changes in the respective standard enthalpies and entropies were mutually interdependent for all eight donors. ΔH ^o=?152+315ΔS ^o(kJ/mol) corresponding to the common value ΔG ^o??152 kJ/mol at 315°K. These mutual interdependencies are possibly the effect of variable conformational states (e.g., the macromolecular compactness) of the cellular receptor pools. This suggestion may furthermore be supported by the correlation traced between ΔH ^o vs the biological age in years of the donors: △H ^°?76.7?1.0 (age)kJ/mol (r = ?0.92) The calculated values of activation enthalpy ΔH ²⁺ kJ/mol and activation entropy ΔS ²⁺ (kJ/mol K) also mutually correlated linearly (r=0.998); the regression line was: △H ²⁺ = ?8.9 + 305△S²⁺ (kJ/mol) corresponding to the common value △H ²⁺ △ ?8.9 (kJ/mol) at 305°K Similarly the activation enthalpy ΔH ²⁺ vs the biological age in years correlated linearly: ΔH ²⁺=67.4?0.73(age) (kJ/mol) (r=?0.76) The range of ΔH ²⁺ studied was from 13.8 to 53.9 kJ/mol with a linearly corresponding range in ΔS ²⁺ from 73 to 205 J/mol K. The thermodynamic data reveal the selenite uptake during the hypothetical standard reaction to be exergonic and endothermic. Critical pH dependencies of the selenite uptake were explained.     

A microcalorimetric study of the reaction catalysed by pyruvate kinase

The enthalpy change for phosphorylation of ADP^3? by PEP^3? catalysed by pyruvate kinase has been determined at 25°C using flow microcalorimetry. Measurements were made at pH 8 in three buffer systems TRIS, TEA and HEPES and also at pH 8.5 in TRIS buffer. The values of ΔH obtained, ?8.75 kJ mol^?1 in TRIS, ?7.3₉ kJ mol^? in TEA and ?6.1₉ kJ mol^?1 in HEPES surprisingly display a dependence on the buffer system used. The enthalpy change was combined with free energy data to calculate the entropy change for the catalysed reaction.     

Kinetics and thermodynamics of the P870+Q−A → P870+Q−B reaction in isolated reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides

The temperature dependences of the P870⁺Q^?_A → P870Q_A and P870⁺Q^?_B → P870Q_B recombination reactions were measured in reaction centers from Rhodopseudomonas sphaeroides. The data indicate that the P870⁺Q^?_B state decays by thermal repopulation of the P870⁺Q^?_A state, followed by recombination. ΔG° for the P870⁺Q^?_A → P870⁺Q^?_B reaction is ?6.89 kJ · mol^?1, while ΔH° = ?14.45 kJ · mol^?1 and ?TΔS° = + 7.53 kJ · mol^?1. The activation ethalpy, $\text{H}{}^3$, for the P870⁺Q^?_A Δ P870⁺Q^?_B reaction is +56.9 kJ · mol^?1, while the activation entropy is near zero. The results permit an estimate of the shape of the potential energy curve for the P870⁺Q^?_A → P870⁺Q^?_B electron transfer reaction.