Kinetic theory of enzymatic reactions in reversed micellar systems

Abstract

A kinetic theory is proposed for enzymatic reactions proceeding in reversed micellar systems in organic solvents, and involving SUbStrdteS capable of partitioning among all pseudophases of the micellar system, i.e. aqueous cores of reversed micelles, micellar membranes and organic solvent. The theory permits determination of true (i.e. with reference to the aqueous phase, where solubilized enzyme is localized) catalytic parameters of the enzyme, provided partition coefficients of the substrate between different phases are known. The validity of the kinetic theory was verified by the example of oxidation of aliphatic alcohols catalyzed by horse liver alcohol dehydrogenase in the system of reversed sodium bis(2-ethylhexyl)sulfosuccinate (AOT, aerosol OT) micelles in octane. In order to determine partition coefficients of alcohols between phases of the micellar system, flow microcalorimetry technique was used. It was shown that in the first approximation, the partition coefficient of the substrate in a simple biphasic system consisting of water and corresponding organic solvent can be used as an estimate for the partition coefficient of the substrate between aqueous and organic solvent phases of the micellar system. True values of the Michaelis constant of alcohols in the micellar system, determined using suggested approach, are equal to those obtained in aqueous solution and differ from apparent values referred to the total volume of the system. The results clearly show that the previously reported shift in the substrate specificity of HLADH, observed on changing from aqueous solution to the system of reversed aerosol OT micelles in octane, is apparent and can be explained on the basis of partitioning effects of alcoholic substrates between phases of the micellar system. Micellar enzymology, which deals with enzymes entrapped in surfactant reversed micelles in orgarlic solvents, has received increasing attention during the last decade (for recent reviews see [l -41). The kinetic description of enzymatic reactions in reversed micellar systems often represents a rather complicated problem because of the microheterogeneity of the micellar medium: micellar solution is composed of tiny surfactantstabilized aqueous microdroplets (with diameter up to 200 nm) suspended in organic solvent. Solubilized enzyme molecules are localized exclusively inside these droplets (micelles), whereas the substrate can be distributed throughout the whole volume of the system. Evidently, the latter fact should be taken into account in kinetic analysis. To allow for substrate partitioning, the pseudophase approach can be used, according to which micellar systems can be regarded as being composed of two phases, namely the phase of organic solvent and a pseudophase of micelles. The pseudophase approach was successfully used for description of the kinetics of nonenzymatic [5] as well as enzymatic [6 -91 reactions in micellar systems. However, the notion that a micellar solution can be viewed as a pseudobiphasic system is to a certain extent on ...

Effect of AOT on enzymatic activity of the organic solvent resistant tyrosinase from Streptomyces sp. REN-21 in aqueous solutions and water-in-oil microemulsions

“…As earlier [8,12], we assume that the main phases of the system are isooctane, water pool, and surfactant layer. Moreover, we assume that the concentration of the substrate in the surfactant layer may be described by the equilibrium of its association with surfactant molecules [12].…”

“…Several attempts at this have been made. Khmelnitsky et al [8] used a pseudophase model of substrate distribution between the organic phase, the water pool, and the interfacial layer to describe the activity of horse liver alcohol dehydrogenase in AOT in octane reverse micelles (at constant w 0 ). Assuming that the enzyme resides exclusively in the aqueous phase, they obtained a relationship between the apparent Michaelis constants K M of the substrate and the volume fractions of all considered microphases.…”

Kinetic behavior of Desulfovibrio gigas aldehyde oxidoreductase encapsulated in reverse micelles

“…The enzyme maximum velocity (V max ) and the Michaelis-Menten constant (K m ) were obtained from a Lineweaver-Burk linearization and reconfirmed by a non-linear regression analysis of the experimental data to the hyperbolic curve defined by a simple Michaelis-Menten equation [17]. Both the enzyme concentration, which is used to calculate the catalytic constant (k cat ¼ V max /[AOR]), and the amount of substrate added in each assay are referred to the total volume of the micelle solution [18,19]. All the kinetic assays were carried out under the same experimental conditions in order to compare the catalytic properties of the enzyme in both systems (reverse micelle and aqueous solutions).…”

“…Although such measurements were reported to give a good estimation of the partition of molecules between the organic and the aqueous pseudophases in a reverse micelle system [19], the micelle membrane, as seen below, may also compete with the other pseudophases to accommodate the aldehyde molecules. Fig.…”

Thermodynamics and modulation of progesterone microcompartmentation and hydrophobic interaction with cytochrome P450XVII based on quantification of local ligand concentrations in a complex multi‐component system

“…The combination of the direct quantitative analysis of ligand partition in subcellular membranes and quantitative analysis of P450-ligand interaction can be expected to lead to a definition of enzyme-ligand binding parameters on the basis of local ligand concentrations. A similar approach has recently been employed for ubiquinol : cytochrome-c reductase [51] and acyl-CoA elongases [52] as well as for liver alcohol dehydrogenase in a reversed micellar system [53].…”