Thermodynamic arguments for temperature-induced cryptic conformational change of human plasma cholinesterase.

The temperature and pressure dependence of the kinetics of the hydrolysis of o-nitrophenylbutyrate by human plasma tetrameric form cholinesterase (EC 3.1.1.8) was studied. The study was carried out on the one hand at atmospheric pressure by spectrophotometry at various temperatures ranging from 0 to 40 degrees C and, on the other hand by high-pressure stopped-flow spectrophotometry at 3.5, 25 and 35 degrees C in the pressure range 10(-3) to 2 kbar. The Arrhenius plot showed a break at 21 +/- 1 degrees C. Kinetic parameters, activation parameters and volume changes are reported. Discontinuities in the thermodynamic quantities obtained from temperature and pressure (up to 0.8 kbar) dependence of hydrolysis rates are discussed; they have been interpreted as the result of a temperature-induced cryptic conformational change of the enzyme at around 20 degrees C. Beyond 1 kbar the kinetics exhibited several complexities: curvature of the progress curves and high positive or negative activation volume changes depending on temperature and substrate concentration. These complex interacting effects between temperature, pressure and substrate concentration are discussed.

Effects of high pressure on the single-turnover kinetics of the carbamylation of cholinesterase.

Pressure, as a perturbing variable, is one of the most powerful tools to investigate the thermodynamic parameters of chemical reactions and to study the mechanism of enzyme-catalyzed reactions. The effect of elevated hydrostatic pressure (up to 0.8 kbar) on the reaction of butyrylcholinesterase with N-methyl-(7-dimethylcarbamoxy)quinolinium was determined under single-turnover conditions at 35 degrees C. The rate of carbamylation was monitored as the accumulation of the fluorescent ion, N-methyl-7-hydroxyquinolinium, in a high-pressure stopped-flow apparatus designed for the assay of fluorescence. Elevated pressure favored formation of the enzyme-substrate complex but inhibited carbamylation of the enzyme. Because a single reaction step was recorded, it was possible to interpret the data obtained under high pressure in the form of Michaelis-Menten equations. From the pressure dependence of the dissociation constant for the enzyme-substrate complex and the rate constant for carbamylation, maximal volume changes accompanying these events were determined. The value for the binding process, delta Vb = -129 ml.mol-1, is too large to be related only to volumetric changes in the active center. Substrate-induced conformational change and change of water structure appear to be the dominant contributions to the overall volume change associated with substrate binding. The large positive activation volume measured (delta V not equal to = 119 ml.mol-1) may also reflect extended structural and hydration changes. At pressures greater than 0.4 kbar, an additional pressure effect, dependent on substrate concentration, occurred in a narrow pressure interval. This effect may have resulted from a substrate-induced pressure-sensitive enzyme conformational state.

Conformational plasticity of butyrylcholinesterase as revealed by high pressure experiments.

The ligand binding and kinetic behaviour of butyrylcholinesterase (EC 3.1.1.8, acylcholine acylhydrolase) from human plasma was studied at 35 degrees C under high hydrostatic pressure. The binding of phenyltrimethylammonium was studied by affinity electrophoresis at various pressures ranging from 10(-3) to 2 kbar. The kinetics of enzyme carbamylation with N-methyl(7-dimethylcarbamoxy)quinolinium iodide was studied in single-turnover conditions up to 1.2 kbar using a high-pressure stopped-flow fluorimeter. Experiments were carried out in different media: 1 mM Tris-HCl (pH 8) with water, water containing 0.1 M lithium chloride and deuterium oxide as solvents. The volume changes (delta V and delta V++) associated with each process were determined from the pressure-dependence of the binding and kinetic constants. Kinetic data show that the binding of substrate to the enzyme leads to a pressure-sensitive enzyme conformational state which cannot accomplish the catalytic act. The pressure-induced inhibitory effect is highly cooperative; it depends on both the nature (charged or neutral) and the concentration of the substrate. Also, large solvent effects indicate that enzyme sensitivity to pressure depends on the solvent structure. This findings suggests that the substrate-dependent pressure effect is modulated by the solvation state of the enzyme.

Recombination of carbon monoxide with hemoglobin after flash photolysis of the carboxyderivative in mixed solvents at subzero temperature.

The kinetics of recombination of carbon monoxide to hemoglobin produced by total flash photolysis of its carboxyderivative are studied at low temperatures (down to --55 degrees C) in mixed hydroalcoholic solvents. The rates are found to be different in two solvents used, namely ethylene glycol/buffer and methanol/buffer; for the former, the rates at subzero temperatures are simply explained by cooling and are consistent with the activation energy as measured in aqueous solution, while those in methanol/buffer show evidence of a specific solvent effect. Values are reported for the rate constants and activation energies in the two solvents.

Catalytic activity of thermolysin under extremes of pressure and temperature: modulation by metal ions.

The catalytic activity of thermolysin (TL), a Zn-dependent neutral protease from Bacillus thermoproteolyticus, has been studied over a wide interval of pressures (1 bar to 4 kbar) and temperatures (20 degreesC to 80 degreesC) by monitoring hydrolysis of a low-molecular-mass substrate, 3-(2-furylacryloyl)-glycyl-L-leucine amide. This reaction shows a very large negative value for the activation volume and, because of that, simultaneous increase in temperature and pressure leads to a significant (up to 40-fold) acceleration of the reaction. At pressures higher than 2-2.5 kbar, the reaction rate starts to decrease due to disactivation of TL. This disactivation is explained in part by pressure-promoted dissociation of zinc ion from the active site and can be inhibited by adding exogenous zinc. Thus, this thermostable protease does not specifically show a higher stability at high pressure in comparison with small mesophilic proteases.

Soman inhibition of butyrylcholinesterase in the presence of substrate: pressure and temperature perturbations.

Irreversible inhibition of butyrylcholinesterase by soman was studied in the presence of the substrate (o-nitrophenyl butyrate). Inhibition was found of the competitive complexing type. Study at different temperatures and pressures showed that the behavior of the enzyme differs from that of the inhibitor-free enzyme. In the absence of inhibitor, enzyme kinetics displayed a non-linear temperature dependence with a break at 21 degrees C. In the presence of a non-inhibitor structural analog of soman (pinacolyl dimethylphosphinate and methyl dimethylphosphinate), the Arrhenius plot break is slightly shifted (18 degrees C). On the other hand, in the presence of soman this break is abolished. The pressure-dependence of the substrate hydrolysis revealed also differences between the native enzyme and the enzyme in the presence of soman: the sign and magnitude of the apparent activation volume (delta V not equal to) were different for the two reactions. Beyond 300 bar, in the presence of soman, a plateau (delta V not equal to approx. 0) was observed over a large pressure range depending on temperature. Such a behavior with respect to temperature and pressure can reflect a soman-induced enzyme conformational state. Thus, temperature and pressure perturbations of the kinetics allow to complete the inhibition scheme of butyrylcholinesterase by soman. Our data suggest that upon soman binding, the enzyme undergoes a long-lived soman-induced-fit conformational change preceding the phosphonylation step. However, an alternative hypothesis according to which the enzyme processes a secondary soman-binding site cannot be ruled out.

Hydrostatic pressure and calcium-induced dissociation of calpains.

The dissociation of mu- and m-calpains was studied by fluorescence spectroscopy under high hydrostatic pressure (up to 650 MPa). Increasing pressure induced a red shift of the tryptophan fluorescence of the calcium-free enzyme. The concentration dependence of the spectral transition was consistent with a pressure-induced dissociation of the subunits. Rising temperature increased the stability of calpain heterodimers and confirmed the predominance of hydrophobic interactions between monomers. At saturating calcium, the spectral transition was not observed for native or iodoacetamide-inactivated calpains, indicating that they were already dissociated by calcium. The reaction volume was about -150 ml mol-1 for both isoforms, and the dissociation constants at atmospheric pressure are approximately 10-12 M and 10-15 M for mu- and m-calpains, respectively. This result indicates a tighter interaction in the isoform that requires higher calcium concentration for activity.

Kinetics of butyrylcholinesterase in reversed micelles under high pressure.

The combined effects of high pressure and reversed micelles have been studied to modulate the catalytic behaviour of butyrylcholinesterase. The purpose of this study was to determine whether the conformational plasticity of the enzyme is altered by entrapment in reversed micelles. The presence of soman, an irreversible inhibitor of cholinesterase was used to bring to the fore a possible modification of the enzyme behaviour in this system under pressure. Results show differences between enzyme in conventional medium and in reversed micelles regarding the mechanism of BuChE catalyzed hydrolysis of acetylthiocholine. In both systems, the enzyme displays a non-Michaelian behaviour with this substrate. In conventional medium the kinetics is multiphasic with an activation phase followed by an inhibition phase at high concentration. In reversed micelles there is inhibition by excess substrate but the activation phase is missing. This behaviour may be the result of a change of the enzyme conformational plasticity when is entrapped in reversed micelles.

Substrate dependence of amiloride- and soman-induced conformation changes of butyrylcholinesterase as evidenced by high-pressure perturbation.

Previous results on butyrylcholinesterase-catalyzed hydrolysis of o-nitrophenylbutyrate in the presence of soman, an irreversible inhibitor of cholinesterases, suggested that reversible binding of soman preceding enzyme phophonylation induced a new enzyme conformational state (E'). The purpose of the present study was to determine whether this effect depends on soman itself or is dependent on the presence and nature of substrate or ligand. First, we examined the effect of amiloride, a reversible cholinesterase effector, upon the butyrylcholinesterase-catalyzed hydrolysis of nitrophenyl esters. The effect of amiloride was found to be dependent on the position ortho or para of the substrate nitro group: amiloride acts as a non-linear reversible activator of p-nitrophenyl ester hydrolysis and as a non-linear reversible inhibitor of o-nitrophenyl ester hydrolysis. Second, the effect of amiloride upon hydrolysis of o/p-nitrophenylbutyrate was also studied under perturbing conditions, i.e., as a function of pressure (1-1600 bar) in the presence and absence of soman. Results show that the effect of reversible soman binding on butyrylcholinesterase activity in the presence of amiloride depends on the position of the substrate nitro group and amiloride concentration. Molecular modelling suggests that the presence of amiloride determines the orientation of ortho- and para-nitrophenyl esters in the active-site. gorge. The nitro group of o-nitrophenylbutyrate interacts with the oxyanion hole via hydrogen bonds and its phenyl ring interacts with amiloride whose heterocycle faces Trp-82. The nitro group of p-nitrophenylbutyrate does not interact with the oxyanion hole but points towards Tyr-332; the phenyl ring of p-nitrophenylbutyrate interacts with amiloride but there is no steric constraint on the acyl chain. Thus, the network of interactions in ternary complexes is tighter with o-nitrophenylbutryate as the substrate. There is no evidence for the existence of amiloride and/or soman-induced E' state when p-nitrophenylbutyrate is the substrate. On the other hand, reversible binding of amiloride and/or soman induces new active conformational states that may be either binary (or ternary) enzyme-ligand complex or new free enzyme conformation resulting from long-lived ligand-induced enzyme conformational change when o-nitrophenylbutyrate is the substrate. These ligand-induced states are stabilized by high pressure.

The powerful high pressure tool for protein conformational studies.

The pressure behavior of proteins may be summarized as a the pressure-induced disordering of their structures. This thermodynamic parameter has effects on proteins that are similar but not identical to those induced by temperature, the other thermodynamic parameter. Of particular importance are the intermolecular interactions that follow partial protein unfolding and that give rise to the formation of fibrils. Because some proteins do not form fibrils under pressure, these observations can be related to the shape of the stability diagram. Weak interactions which are differently affected by hydrostatic pressure or temperature play a determinant role in protein stability. Pressure acts on the 2 degrees, 3 degrees and 4 degrees structures of proteins which are maintained by electrostatic and hydrophobic interactions and by hydrogen bonds. We present some typical examples of how pressure affects the tertiary structure of proteins (the case of prion proteins), induces unfolding (ataxin), is a convenient tool to study enzyme dissociation (enolase), and provides arguments to understand the role of the partial volume of an enzyme (butyrylcholinesterase). This approach may have important implications for the understanding of the basic mechanism of protein diseases and for the development of preventive and therapeutic measures.

A stopped-flow study of the reaction of cytochrome c peroxidase with hydroperoxides.

Transient kinetic measurements show that cytochrome c peroxidase reacts with excess of hydroperoxides to produce compound ES in two phases. The activation energies for the fast and slow phases are calculated to be 6.3 and 20.5 kcal X mol-1, respectively. The fast phase is assigned to the reaction of native active (pulsed) cytochrome c peroxidase with peroxides, whereas the slow phase is due to the presence of an inactive (aged, resting) enzyme. As the active species is exhausted, the equilibrium between the active and inactive enzymes is shifted by a slow conformational change to replenish the active enzyme. Since the rate-limiting step of the reaction of the inactive enzyme with peroxides is the conformation change, the overall reaction rate is independent of the nature and concentration of peroxides.

High-pressure stabilization of alpha-chymotrypsin entrapped in reversed micelles of aerosol OT in octane against thermal inactivation.

alpha-Chymotrypsin (CT) solubilized in reversed micelles of sodium bis-(2-ethylhexyl)-sulfosuccinate (AOT) undergoes thermal inactivation and the enzyme stability decreases significantly when temperature increases (25-40 degrees C). The half-life of CT in micelles shows a bell-shaped dependence on the degree of hydration of AOT (wo) analogous to the previously obtained dependence on wo for the enzyme activity. The optima of catalytic activity and thermal stability have been observed under conditions where the diameter of the inner aqueous cavity of the micelle is close to the size of the enzyme molecule (wo = 10). Application of high hydrostatic pressure in the range of 1-1500 atm (bar) stabilizes CT against thermal inactivation at all hydration degrees (wo) from 7 to 20; the stabilization effect is most pronounced under the experimental conditions being far from the optimum for catalytic activity.

New trends in cryoenzymology : II. - Aqueous solutions of enzymes in apolar solvents.

In order to set up new procedures to investigate enzyme systems at subzero temperatures in pure aqueous media, we used micromicellar solutions which are homogeneous, optically transparent and of low viscosity in that range of temperatures. The preparation and the main properties of such solutions are described along with the behavior of enzyme-substrate intermediates. A critical discussion of results permits to examine advantages as well as limitations of this very promising procedure.

New trends in cryoenzymology: I.-Supercooled aqueous solutions.

A water in oil emulsion technique is proposed to investigate enzyme catalyzed reactions at sub-zero temperatures in the supercooled liquid state to avoid some reversible effects of the usual cosolvents on kinetics. Some results are listed: potentialities and technical problems of the procedure are discussed.

Preliminary studies on quinoprotein glucose dehydrogenase under extreme conditions of temperature and pressure.

The kinetics of the reduction of the quinoprotein glucose dehydrogenase by substrate were studied as a function of 3 parameters: pressure (1-1000 bar), temperature (down to -25 degrees C) and solvent (water and 40% dimethyl sulfoxide, DMSO) using a high-pressure low-temperature stopped-flow apparatus. A 2-step formation of the reduced enzyme by its substrate (xylose), was observed. A rapid equilibrium described by the constant K1 was followed by a slower process described by the constants k2 and k-2. By using the transition state theory, the thermodynamic quantities delta V (activation volumes) were determined for these various kinetics constants under different experimental conditions. The results are discussed in terms of conformational change and solvation effect on the protein shell, and compared with results obtained for other systems as the 2-step formation of horseradish peroxidase compound I.

New trends in cryoenzymology: probing the functional role of protein dynamics by single-step kinetics.

Cryoenzymology was initially used to slow down enzyme-catalyzed reactions so as to stabilize intermediates for further study. During the course of this early work, it became clear that cryoenzymology could be extended to other ends and some of these are described. First, the use of a cryosolvent on its own (or together with temperature) as a perturbant has allowed a resolution of the substrate binding steps of certain enzymes (myosin, D-amino acid oxidase, peroxidase and cytochrome P450). Second, by the use of cryosolvent and temperature, coupled with the classical physico-chemical perturbants, one can selectively modulate the various steps of an enzyme pathway. This approach can lead to an understanding of the mechanism of enzyme regulation. Finally, by carrying out experiments over a wide range of temperatures (-30 degrees C- +30 degrees C) and pressure (up to several kbars) in specially constructed fast reaction equipment, one can study the thermodynamic properties of the individual rate constants describing the interconversions of reaction intermediates. Experiments with creatine kinase, cytochrome P450 and peroxidase are described. The thermodynamic parameters delta H, delta G, delta S and delta V are thus measured and when this is done under different solvent conditions one can, at least within the theories available, attempt an approach to the problem of protein dynamics.

Fast photochemical reactions of cytochrome P450 at subzero temperatures.

Several reactions of the cytochrome P450 multi-step cycle have been studied by fast light activation combined with subzero temperatures. A flash device was adapted to an Aminco-Chance DW 2 spectrophotometer equipped for subzero temperature thermostatisation. The first electron can be introduced into the cycle by non specific reducing agents such as reduced flavin mononucleotide (FMNH2) or methylviologen radical (MV.). This first reduction remains a fast process even at subzero temperatures. The oxy-compound Fe2+-O2 can thus be formed either directly from Fe2+ or via the photodissociation of the carboxy-ferro adduct. Fe2+-O2 is stable at subzero temperatures towards spontaneous autoxidation as well as further reduction by FMNH2 or MW.. In addition, the recombination of CO after flash photodissociation of Fe2+-CO was used to study in more details the specific behaviors of the purified microsomal cytochrome.

Organic solvents and temperature effects on desorption from immunoadsorbents. DNP-BSA anti-DNP as a model.

Conditions for desorbing a DNP carrier from its immunoadsorbent were studied in various hydro-organic media at different pHs and temperatures, including the sub-zero range. It appeared that DNP--anti-DNP interaction is the result of a balance between various bonds and the best result (95% yield) was obtained in hydro-organic solvent at high pH and +30 degrees C.

The effects of organic solvents and temperature on the desorption of yeast 3-phosphoglycerate kinase from immunoadsorbent.

Physicochemical parameters governing the elution of yeast 3-phosphoglycerate kinase from its immunoadsorbent were studied. Non-denaturing elution conditions were determined (alkali medium containing 50%, v/v, ethylene glycol) and the method was applied to one-step isolation of enzyme from a crude yeast preparation.

Inductive and repressive effects of rifampicin on rabbit liver microsomal cytochrome P-450.

New Zealand White rabbits were treated with rifampicin at a dose of 50 mg/kg for 4 days. The total amount of microsomal hepatic cytochrome P-450 was not modified in treated, with respect to control, animals. However, further studies involving SDS-PAGE analysis, monooxygenase activity measurements and radial immunodiffusion assays indicated that rifampicin strongly affects the level of two P-450 isoenzymes. An LM3 form was induced; this form, apparently associated with erythromycine demethylase activity and hydroxylation of progesterone preferentially in position 6 beta, was shown to be immunologically and functionally different from LM3a and LM3b. On the other hand, an LM4 form, typically induced by beta-naphthoflavone, was repressed. The concomitant inductive/repressive effect of rifampicine on two cytochrome P-450 isoenzymes makes this drug a very atypical inducer, at least in the rabbit.