Activation Volumes ΔV Research Articles

Pressure accelerates the circadian clock of cyanobacteria

Although organisms are exposed to various pressure and temperature conditions, information remains limited on how pressure affects biological rhythms. This study investigated how hydrostatic pressure affects the circadian clock (KaiA, KaiB, and KaiC) of cyanobacteria. While the circadian rhythm is inherently robust to temperature change, KaiC phosphorylation cycles that were accelerated from 22 h at 1 bar to 14 h at 200 bars caused the circadian-period length to decline. This decline was caused by the pressure-induced enhancement of KaiC ATPase activity and allosteric effects. Because ATPase activity was elevated in the CI and CII domains of KaiC, while ATP hydrolysis had negative activation volumes (ΔV≠), both domains played key roles in determining the period length of the KaiC phosphorylation cycle. The thermodynamic contraction of the structure of the active site during the transition state might have positioned catalytic residues and lytic water molecules favourably to facilitate ATP hydrolysis. Internal cavities might represent sources of compaction and structural rearrangement in the active site. Overall, the data indicate that pressure differences could alter the circadian rhythms of diverse organisms with evolved thermotolerance, as long as enzymatic reactions defining period length have a specific activation volume.

Complexation of [Gd(DTTA-Me)(H2O)2](-) by Fluoride and Its Consequences to Water Exchange.

The displacement of water molecule(s) from the inner coordination sphere of [Gd(DTTA–Me)(H2O)2]− (DTTA = ethylenetriamine-N,N,N″,N″-tetraacetate) by fluoride has been studied by multinuclear NMR relaxation (1H, 17O, 19F) and DFT calculations. Fluoride anions can replace only one of the coordinated water molecules. The thermodynamic stability constant (KGdLF,2980 = 11.6 ± 0.3) and thermodynamic parameters characterizing the formation of [Gd(DTTA–Me)(H2O)F]2– were determined (ΔH0 = +6.3 ± 0.1 kJ mol–1; ΔS0 = +41.5 ± 3.4 J mol–1 K–1; ΔV0 = +4.5 ± 1.2 cm3 mol–1). Fluoride binding causes a marked acceleration of the water exchange, which is seven times faster for [Gd(DTTA–Me)(H2O)F]2– (kex,1298 = 177 × 106 s–1) than for [Gd(DTTA–Me)(H2O)2]− (kex,2298 = 24.6 × 106 s–1). Water exchange on both compounds is faster than formation of the fluoride complex. The analysis of the Gd–Owater distances, electron density, and electron localization function (ELF) at the bond critical points using DFT calculations reveals that F– binding weakens the Gd–Owater bonds, thereby facilitating the departure of the coordinated water molecule following a dissociative mechanism. The water exchange on both Gd(DTTA–Me) complexes follow dissociative reaction pathways as shown by the positive activation volumes ΔV⧧ = +8 ± 2 cm3 mol–1 and +15 ± 4 cm3 mol–1 for the bis-aqua complex and the monofluoro complex, respectively.

Do TFSA Anions Slither? Pressure Exposes the Role of TFSA Conformational Exchange in Self-Diffusion.

Multinuclear ((1)H, (2)H, and (19)F) magnetic resonance spectroscopy techniques as functions of temperature and pressure were applied to the study of selectively deuterated 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)amide (EMIM TFSA) ionic liquid isotopologues and related ionic liquids. For EMIM TFSA, temperature-dependent (2)H T1 data indicate stronger electric field gradients in the alkyl chain region compared to the imidazolium ring. Most significantly, the pressure dependences of the EMIM and TFSA self-diffusion coefficients revealed that the displacements of the cations and anions are independent, with diffusion of the TFSA anions being slowed much more by increasing pressure than for the EMIM cations, as shown by their respective activation volumes (28.8 ± 2.5 cm(3)/mol for TFSA vs 14.6 ± 1.3 cm(3)/mol for EMIM). Increasing pressure may lower the mobility of the TFSA anion by hindering its interconversion between trans and cis conformers, a process that is coupled to diffusion according to published molecular dynamics simulations. Measured activation volumes (ΔV(‡)) for ion self-diffusion in EMIM bis(fluoromethylsulfonyl)amide and EMIM tetrafluoroborate support this hypothesis. In addition, (2)H T1 data suggest increased ordering with increasing pressure, with two T1 regimes observed for the MD3 and D2 isotopologues between 0.1-100 and 100-250 MPa, respectively. The activation volumes for T1 were 21 and 25 cm(3)/mol (0-100 MPa) and 11 and 12 cm(3)/mol (100-250 MPa) for the MD3 and D2 isotopologues, respectively.

The solvent effect on two competing reaction mechanisms involving hypervalent iodine reagents (λ3‐iodanes): Facing the limit of the stationary quantum chemical approach

Trifluoromethylation of acetonitrile with 3,3-dimethyl-1-(trifluoromethyl)-1λ(3),2- benziodoxol is assumed to occur via reductive elimination (RE) of the electrophilic CF3-ligand and MeCN bound to the hypervalent iodine. Computations in gas phase showed that the reaction might also occur via an SN2 mechanism. There is a substantial solvent effect present for both reaction mechanisms, and their energies of activation are very sensitive toward the solvent model used (implicit, microsolvation, and cluster-continuum). With polarizable continuum model-based methods, the SN2 mechanism becomes less favorable. Applying the cluster-continuum model, using a shell of solvent molecules derived from ab initio molecular dynamics (AIMD) simulations, the gap between the two activation barriers ( ΔΔG‡) is lowered to a few kcal mol(-1) and also shows that the activation entropies (ΔS‡) and volumes (ΔV‡) for the two mechanisms differ substantially. A quantitative assessment of ΔΔG‡ will therefore only be possible using AIMD. A natural bond orbital-analysis gives further insight into the activation of the CF3-reagent by protonation.

Temperature and Pressure Effects on C–H Abstraction Reactions Involving Compound I and II Mimics in Aqueous Solution

The presented results cover a comparative mechanistic study on the reactivity of compound (Cpd) I and II mimics of a water-soluble iron(III) porphyrin, [meso-tetrakis(2,4,6-trimethyl-3-sulfonatophenyl)porphinato]iron(III), Fe(III)(TMPS). The acidity of the aqueous medium strongly controls the chemical nature and stability of the high-valent iron(IV) oxo species. Reactivity studies were performed at pH 5 and 10, where the Cpd I and II mimics are stabilized as the sole oxidizing species, respectively. The contributions of ΔH(‡) and ΔS(‡) to the free energy of activation (ΔG(‡)) for the oxidation of 4-methoxybenzaldehyde (4-MB-ald), 4-methoxybenzyl alcohol (4-MB-alc), and 1-phenylethanol (1-PhEtOH) by the Cpd I and II mimics were determined. The relatively large contribution of the ΔH(‡) term in comparison to the -TΔS(‡) term to ΔG(‡) for reactions involving the Cpd II mimic indicates that the oxidation of selected substrates by this oxidizing species is clearly an enthalpy-controlled process. In contrast, different results were found for reactions with application of the Cpd I mimic. Depending on the nature of the substrate, the reaction at room temperature can be entropy-controlled, as found for the oxidation of 4-MB-alc, or enthalpy-controlled, as found for 1-PhEtOH. Importantly, for the first time, activation volumes (ΔV(‡)) for the oxidation of selected substrates by both reactive intermediates could be determined. Positive values of ΔV(‡) were found for reactions with the Cpd II mimic and slightly negative ones for reactions with the Cpd II mimic. The results are discussed in the context of the oxidation mechanism conducted by the Cpd I and II mimics.

Pyridine‐Based Lanthanide Complexes Combining MRI and NIR Luminescence Activities

A series of novel triazole derivative pyridine-based polyamino-polycarboxylate ligands has been synthesized for lanthanide complexation. This versatile platform of chelating agents combines advantageous properties for both magnetic resonance (MR) and optical imaging applications of the corresponding Gd(3+) and near-infrared luminescent lanthanide complexes. The thermodynamic stability constants of the Ln(3+) complexes, as assessed by pH potentiometric measurements, are in the range log K(LnL)=17-19, with a high selectivity for lanthanides over Ca(2+), Cu(2+), and Zn(2+). The complexes are bishydrated, an important advantage to obtain high relaxivities for the Gd(3+) chelates. The water exchange of the Gd(3+) complexes (k(ex)(298)=7.7-9.3×10(6) s(-1)) is faster than that of clinically used magnetic resonance imaging (MRI) contrast agents and proceeds through a dissociatively activated mechanism, as evidenced by the positive activation volumes (ΔV(≠)=7.2-8.8 cm(3) mol(-1)). The new triazole ligands allow a considerable shift towards lower excitation energies of the luminescent lanthanide complexes as compared to the parent pyridinic complex, which is a significant advantage in the perspective of biological applications. In addition, they provide increased epsilon values resulting in a larger number of emitted photons and better detection sensitivity. The most conjugated system PheTPy, bearing a phenyl-triazole pendant on the pyridine ring, is particularly promising as it displays the lowest excitation and triplet-state energies associated with good quantum yields for both Nd(3+) and Yb(3+) complexes. Cellular and in vivo toxicity studies in mice evidenced the non-toxicity and the safe use of such bishydrated complexes in animal experiments. Overall, these pyridinic ligands constitute a highly versatile platform for the simultaneous optimization of both MRI and optical properties of the Gd(3+) and the luminescent lanthanide complexes, respectively.

High‐pressure analysis of a hammerhead ribozyme from Chrysanthemum chlorotic mottle viroid reveals two different populations of self‐cleaving molecule

The activity of the full-length hammerhead ribozyme requires a tertiary interaction between its distal loops leading to the closure of the molecule and its stabilization in the active conformation. In this study, the conformational changes accompanying the cis-cleavage reaction of Chrysanthemum chlorotic mottle viroid hammerhead ribozyme were investigated by high-pressure experiments on the complete cleavage reaction. Two activation volumes (ΔV(≠)) were measured, pointing to the presence of two different populations of molecules corresponding to fast-cleaving and slow-cleaving ribozymes in the reaction mixture. The fast population, with a small ΔV(≠) of 2.6 mL·mol(-1), most likely represents molecules in the near-active conformation, whereas the slow population, with a larger ΔV(≠) of 11.6 mL·mol(-1 , represents molecules that need a larger conformational change to induce activity. In addition, pH-dependence experiments suggest that the group whose deprotonation is required for activity intervenes in the formation of the transition state or in the chemistry of the reaction, but not in the conformational change that precedes it.

Laboratory measurements of electrical conductivities of hydrous and dry Mount Vesuvius melts under pressure

Quantitative interpretation of MT anomalies in volcanic regions requires laboratory measurements of electrical conductivities of natural magma compositions. The electrical conductivities of three lava compositions from Mount Vesuvius (Italy) have been measured using an impedance spectrometer. Experiments were conducted on both glasses and melts between 400 and 1300°C, at both ambient pressure in air and high pressures (up to 400 MPa). Both dry and hydrous (up to 5.6 wt % H2O) melt compositions were investigated. A change of the conduction mechanism corresponding to the glass transition was systematically observed. The conductivity data were fitted by sample‐specific Arrhenius laws on either side of Tg. The electrical conductivity increases with temperature and is higher in the order tephrite, phonotephrite to phonolite. For the three investigated compositions, increasing pressure decreases the conductivity, although the effect of pressure is relatively small. The three investigated compositions have similar activation volumes (ΔV = 16–24 cm3 mol−1). Increasing the water content of the melt increases the conductivity. Comparison of activation energies (Ea) from conductivity and sodium diffusion and use of the Nernst‐Einstein relation allow sodium to be identified as the main charge carrier in our melts and presumably also in the corresponding glasses. Our data and those of previous studies highlight the correlation between the Arrhenius parameters Ea and σ0. A semiempirical method allowing the determination of the electrical conductivity of natural magmatic liquids is proposed, in which the activation energy is modeled on the basis of the Anderson‐Stuart model, σ0 being obtained from the compensation law and ΔV being fitted from our experimental data. The model enables the electrical conductivity to be calculated for the entire range of melt compositions at Mount Vesuvius and also satisfactorily predicts the electrical response of other melt compositions. Electrical conductivity data for Mount Vesuvius melts and magmas are slightly lower than the electrical anomaly revealed by MT studies.

Thermodynamic and Kinetic Characterization of Porous Graphitic Carbon in Reversed‐Phase Liquid Chromatography

The thermodynamics and kinetics of solute transfer on porous graphitic carbon (PGC) are studied for two series of aromatic hydrocarbons, alkylbenzenes and methylbenzenes. The retention behavior is characterized as a function of temperature, pressure, length of alkyl chain, and number and position of methyl substituents. In the thermodynamic studies, the retention factor (k) increases with an increase in the length of alkyl chain or number of methyl substituents. The data indicate that increases in the number of methylene and methyl substituents result in more negative changes in molar enthalpy (ΔHsm). Hence, the transition from mobile to stationary phase is a more energetically favorable, exothermic process with each methylene or methyl group added. The data also show that the change in molar volume (ΔVsm) is close to zero, which is consistent with an adsorption mechanism on PGC. Enthalpy‐entropy compensation is observed for both homologous series, and the compensation temperature suggests that the retention mechanism is distinctly different for the methylene and methyl groups. In the kinetic studies, the rate of solute transfer increases with an increasing number of methylene and methyl groups. The activation enthalpy from the stationary phase to transition state (ΔH‡s) is found to increase with an increasing number of methylene and methyl groups. But the activation enthalpy from the mobile phase to transition state (ΔH‡m) is much smaller than ΔH‡s and, at the same time, is very similar for all the solutes. The activation volumes (ΔV‡s and ΔV‡m) are very close to zero, again owing to the adsorption mechanism on PGC. These results demonstrate that the retention mechanism for PGC, which is based on adsorption, is different from that for octadecylsilica, the most widely used stationary phase in liquid chromatography.

Distinct Water-Exchange Mechanisms for Trinuclear Transition-Metal Clusters

Mechanisms for water exchange from the bioxo-capped M-M-bonded trinuclear clusters, [M3(mu3-O)2(mu-O2CCH3)6(OH2)3]2+ [M = Mo(IV) and W(IV)], were investigated using high-pressure 17O NMR and compared to our previous work on a similar Rh(III) trimer. Reaction rates decrease by more than a factor of 2 when pressure is increased from 6 to 250 MPa for the Mo(IV) trimer, while exchange rates increase by less than a factor of 1.2 (10-229 MPa) for the W(IV) trimer. From the pressure dependence of the reaction rate, activation volumes (DeltaV()) were calculated to be DeltaV() = +8.0 (+/-0.4) cm(3) mol(-1) and DeltaV = -1.9 (+/-0.2) cm(3) mol(-1) for the Mo(IV) cluster and W(IV) cluster, respectively, which is the largest difference ( approximately 10 cm(3) mol(-1)) in activation volumes for any pair of 4d-5d (and 3d-4d) transition metal species located within the same group of the periodic table. If we interpret these activation volumes in terms of Swaddle's semiempirical model, which he established for simple octahedral monomers (Associative (A) = DeltaV approximately -13; Interchange (I) = DeltaV approximately 0; or Dissociative (D) = DeltaV approximately +13), our results suggest that water exchange follows a dissociative-interchange (Id) mechanism for the Mo(IV) cluster and an associative-interchange (Ia) activation mode for the W(IV) trimer. These volumes exhibit a unique changeover in the water-exchange mechanism despite considerable similarities in molecular structure and reactivity. This changeover could provide a standard for computational simulations of ligand-exchange pathways in molecules that are more complicated than monomers.

Oxide-Ion Transport in Gadolinium Zirconate - Titanates under High Pressure

ABSTRACTThe oxide-ion conduction of gadolinium zirconate - titanates, Gd2(Ti1−xZrx)2O7 (GTZ), has been investigated under high pressure in the range of 2 to 6 GPa with the aid of a cubic-anvil-type apparatus. For these pyrochlore compounds, the oxide-ion conductivity at elevated temperatures in the range of 750 to 960 °C decreased with increasing pressure, indicating the presence of a positive activation volume, ΔV. The activation volumes ΔV of GZ (x = 1.0) and GTZ (x = 0.95) were 2.0 and 1.3 cm3/mol, respectively, at 750 °C and decreased with increasing temperature.

Can octahedral t2g6 complexes substitute associatively? The case of the isoelectronic ruthenium(II) and rhodium(III) hexaaquaions.

For the low-spin t2g6 Ru(OH2)6(2+) (delta V++ = -0.4 cm3 mol-1) and Rh(OH2)6(3+) (delta V++ = -4.2 cm3 mol-1) hexaaquaions, the respective Id and Ia water exchange mechanisms had been assigned, mainly on the basis of activation volumes delta V++ and entering ligands effects for water substitution. For Ru(II) the near-zero delta V++ was supposed to be due to the compensation between a positive contribution (the loss of a water molecule) and a negative one (the contraction of the bonds of the five spectator ligands at the transition state). Recently, it has been suggested that Rh(III), because of its higher positive charge, could promote further spectator ligands bond contraction sufficient to change the sign of delta V++ to a negative value. If true, this would be an example of limitation in the use of delta V++ for a direct diagnosis of the mechanism. Quantum chemical calculations including hydration effects show that the activation energies for the water exchange on Rh(OH2)6(3+) via the Ia (114.8 kJ mol-1) and the D pathways is 21.8 kJ mol-1 in favor of the former. In the case of Ru(OH2)6(2+) all attemps to compute a transition state for an interchange mechanism failed, but the calculated delta E++ for the D mechanism (71.9 kJ mol-1) is close to both experimental delta G298++ and delta H298++ values. The calculated delta sigma d(M-O) values of -0.53 A for rhodium(III) and +1.25 A for ruthenium(II) agree with the experimented delta V++ values and suggest Ia and D (or Id) mechanisms, respectively. In the case of Ru(OH2)6(2+) the shortening of the bonds of the five spectator ligands to reach the transition states corresponds to a volume change of -1.7 cm3 mol-1. For Rh(OH2)6(3+) these spectator ligands' volume decrease is much smaller (maximum of -0.8 cm3 mol-1) and the bond lengths of the two exchanging ligands at the transition state are characteristic of an interchange pathway with a small "a" character. Because of the strong RhIII-O bonds, water exchange on Rh(OH2)6(3+) proceeds via the Ia pathway with retention of the configuration, whereas the same reaction of Ru(OH2)6(2+), which has considerably weaker RuII-O bonds, follows the Id or the D mechanism.

Unusual Pressure Effects on Ligand Rebinding to the Human Myoglobin Leucine 29 Mutants

Using high pressure flash photolysis, we revealed that the side chain of Leu(29) controls the reaction volume of the ligand migration process in myoglobin, which is the primary factor for the unusual activation volume of ligand binding in some Leu(29) mutants. As we previously reported (Adachi, S., Sunohara, N., Ishimori, K., and Morishima, I. (1992) J. Biol. Chem. 267, 12614-12621), CO bimolecular rebinding in the L29A mutant was unexpectedly decelerated by pressurization, suggesting that the rate-determining step is switched to ligand migration. However, very slow CO bimolecular rebinding of the mutants implies that bond formation is still the rate-determining step. To gain further insights into effects of the side chain on ligand binding, we prepared some new Leu(29) mutants to measure the CO and O(2) rebinding reaction rates under high hydrostatic pressure. CO bimolecular rebinding in the mutants bearing Gly or Ser at position 29 was also decelerated upon pressurization, resulting in apparent positive activation volumes (DeltaV), as observed for O(2) binding. Based on the three-state model, we concluded that the increased space available to ligands in these mutants enhances the volume difference between the geminate and deoxy states (DeltaV(32)), which shifts the apparent activation volume to the positive side, and that the apparent positive activation volume is not due to contribution of the ligand migration process to the rate-determining step.

Pressure-induced activation and pressure-tolerance of mutated thermolysin at 119

The pressure activation of thermolysin substitution at the 119th site was studied for four mutants and the wild type (Q119Q. Q119N, Q119R, Q119E and Q119D). The highest activation recorded over 30 fold and the activation volumes (ΔV‡) were about -75ml/mol for Q119Q, Q119N and Q119R, while Q119E and Q119D showed only a10 fold activation and ΔV‡ of around -6Oml/mol. The pressure-tolerance of these enzymes were investigated through the in situ observation of their intrinsic fluorescence. Q119E and Q119D showed smaller ΔG app and ΔG app of transition than the wild type.

Phantom Activation Volumes

Activation volumes ΔV≠ are presently obtained by measuring the effect of pressure on the reaction rate. It is implicitly assumed that the entire response of rate to pressure is volume related, i.e., acceleration by high pressure reveals shrinkage as the reactants progress to the transition state, and vice versa. However, we now demonstrate that high pressure accelerates some bond-making reactions in an additional, nonvolume-related way, through its elevation of solvent viscosity. Diels−Alder reactions, 1,3-dipolar cycloadditions, and Claisen rearrangements are accelerated by rising viscosity and are therefore subject to viscosity-associated acceleration at raised pressures. This gives rise to the illusion of volume shrinkage along the reaction coordinate toward the transition state, which we term phantom ΔV≠. Thus the true ΔV≠ for these reactions, while negative, is less negative than previously believed. Corrections in ΔV≠, calculated from experimental rate−viscosity plots, range up to 61%.

Pressure Dependence of the Intramolecular Electron Transfer Reaction in Myoglobin Reinvestigated

The activation volumes (ΔV≠) for intramolecular electron transfer (ET) reactions in Ru-modified Zn-porphyrin (ZnP) substituted myoglobins (Ru−ZnMb) have been determined to investigate the pressure effects on the redox potentials and donor−acceptor distance (D−A distance) for the ET reaction. Three Ru−ZnMbs, in which D−A distances for the ET reactions are 12.7 A (His48Mb), 15.5 A (His83Mb), and 19.3 A (His81Mb), were constructed. The activation volumes for the forward ET reactions (ΔV≠f) were −1.6 (His83Mb), +3.7 (His81Mb), and +6.5 cm3 mol-1 (His48Mb). We also measured the pressure dependence of the back ET reactions (from Ru2+ complex to ZnP+), showing that the back ET reactions exhibited negative activation volumes (ΔV≠b) for all of the Ru−ZnMbs: −11, −5.3, and −6.2 cm3 mol-1 for His83Mb, His81Mb, and His48Mb, respectively. On the basis of these activation volumes, the pressure dependence of the redox potentials, (∂ΔG°/∂P)T, was estimated as about 2.94 × 10-4 eV MPa-1, regardless of the position of the ...

The Effect of Pressure on Retro Diels–Alder Reactions

The activation and reaction volumes (ΔV#/ΔV) were determined for the retro Diels–Alder reactions of the parent dihydrobarrelene 1a, its 2-cyano derivative 1b, the exo and endo Diels–Alder adducts of maleic anhydride to naphthalene exo-, endo-4, and the exo and endo Diels–Alder adducts of N-phenylmaleic imide to 6,6-dimethylfulvene exo-, endo-8 from the pressure dependence of the rate constants and the partial molar volumes (V) of reactants and products at various temperatures. The cleavage of exo-4, endo-4, and exo-8 are slightly accelerated by pressure showing negative volumes of activation (ΔV# < 0) whereas the others are slightly retarded (ΔV# > 0). From the analysis of the volume data including the van der Waals volumes (Vw) one can conclude that the packing coefficients of the pericyclic transition states are equal to or even larger than those of the corresponding Diels–Alder adducts. This finding may be explained with the restriction of the degrees of freedom in the transition states leading to a contraction of the expansion volume.

Hydration change during the aging of phosphorylated human butyrylcholinesterase: importance of residues aspartate-70 and glutamate-197 in the water network as probed by hydrostatic and osmotic pressures

Wild-type human butyrylcholinesterase (BuChE) and Glu-197-->Asp and Asp-70-->Gly mutants (E197D and D70G respectively) were inhibited by di-isopropyl phosphorofluoridate under standard conditions of pH, temperature and pressure. The effect of hydrostatic and osmotic pressures on the aging process (dealkylation of an isopropyl chain) of phosphorylated enzymes [di-isopropylated (DIP)-BuChE] was investigated. Hydrostatic pressure markedly increased the rate of aging of wild-type enzyme. The average activation volume (DeltaV( not equal)) for the dealkylation reaction was -170 ml/mol for DIP wild-type BuChE. On the other hand, hydrostatic pressure had little effect on the aging of the DIP mutants (DeltaV( not equal)=-2.6 ml/mol for E197D and -2 ml/mol for D70G), suggesting that the transition state of the aging process was associated with an extended hydration and conformational change in wild-type BuChE, but not in the mutants. The rate of aging of wild-type and mutant enzymes decreased with osmotic pressure, allowing very large positive osmotic activation volumes (DeltaV not equal osm) to be estimated, thus probing the participation of water in the aging process. Molecular dynamics simulations performed on the active-site gorge of the wild-type DIP adduct showed that the isopropyl chain involved in aging was highly solvated, supporting the idea that water is important for stabilizing the transition state of the dealkylation reaction. Wild-type BuChE was inhibited by soman (pinacolyl methylphosphonofluoridate). Electrophoresis performed under high pressure [up to 2.5 kbar (1 bar=10(5) Pa)] showed that the soman-aged enzyme did not pass through a pressure-induced, molten-globule transition, unlike the native wild-type enzyme. Likewise, this transition was not seen for the native E197D and D70G mutants, indicating that these mutants are resistant to the penetration of water into their structure. The stability energetics of native and soman-aged wild-type BuChE were determined by differential scanning calorimetry. The pH-dependence of the midpoint transition temperature of endotherms indicated that the high difference in stabilization energy between aged and native BuChE (DeltaDeltaG=23.7 kJ/mol at pH 8.0) is mainly due to the salt bridge between protonated His-438 and PO(-), with pK(His-438)=8.3. A molecular dynamics simulation on the MIP adduct showed that there is no water molecule around the ion pair. The 'hydrostatic versus osmotic pressure' approach probed the importance of water in aging, and also revealed that Asp-70 and Glu-197 are the major residues controlling both the dynamics and the structural organization of the water/hydrogen-bond network in the active-site gorge of BuChE. In wild-type BuChE both residues function like valves, whereas in the mutant enzymes the water network is slack, and residues Gly-70 and Asp-197 function like check valves, i.e. forced penetration of water into the gorge is not easily achieved, thereby facilitating the release of water.

Structural and hydration changes in the active site gorge of phosporhylated butyrylcholinesterase accompanying the aging process

Wild-type (wt) butyrylcholinesterase (BuChE) and the E197D and D70G mutants were inhibited by diisopropylfluorophosphate (DFP) or soman under standard conditions of pH, temperature and pressure. The effect of hydrostatic and osmotic pressures on the aging process of DFP-phosphorylated enzymes (diisopropylphosphoryl-BuChE (DIP-BuChE)) was investigated. Hydrostatic pressure strongly increased the rate of aging of wt enzyme. The activation volumes (ΔV≠) for the dealkylation reaction was −150 ml/mol for DIP-wtBuChE. On the other hand, pressure had little effect on the aging of the DIP-E197D mutant and no effect on the DIP-D70G mutant, indicating that the transition state of the aging reaction (dealkylation of an isoproxy chain) was associated with an extended conformation/hydration change in wtBuChE but not in mutants. The rate of aging decreased with osmotic pressure, supporting the idea that water is important for stabilizing the transition state. Molecular dynamics simulations were performed on the wtDIP adduct to relate the kinetic data to hydration changes in the enzyme active site gorge. The pH dependence of the melting temperature (Tm) of native and soman-wtBuChE, as determined by differential scanning calorimetry (DSC), indicated that the stabilization energy of aged BuChE is mainly due to the salt bridge between protonated H438 and PO−, with pKH438=8.3. Electrophoresis under high pressure up to 2.5 kbar showed that aged wtBuChE did not undergo pressure-induced molten globule transition unlike the native enzyme. This transition was not seen for the mutant enzymes, indicating that mutants are resistant to the penetration of water into their structure. Our results support the conclusion that D70 and E197 are major residues for the water/H-bond network dynamics in the active site gorge of BuChE, both residues acting like valves. In mutant enzymes, mutated residues function like check valves: forced penetration of water in the gorge is difficult, release of water is facilitated.

Activation volumes for the quenching of the first electronically excited singlet state of oxygen by toluene molecules in pure toluene and four different solvents

Second-order rate constants k D, of the quenching of singlet oxygen in its a 1Δ g state by toluene molecules in bulk solvent and four different solvent environments have been determined with the pressure dependence up to 1200 bar, using time-resolved phosphorescence. Experimental activation volumes ΔV ‡ exp were analysed taking into account contact complex formation as the initial step. The reaction volume of the formation of the contact complex Δ V C was evaluated using the hard-sphere model of Yoshimura and Nakahara. The data indicate that Δ V C is the main part of ΔV ‡ exp . A correlation was found between the k D values at 1 bar and the radial distribution function, as expected of the theory of Einwohner and Alder. The quenching rate constants for benzene and cyclohexane at normal pressure are in agreement with this correlation.