The dependence of the catalytic activities of a-chymotrypsin and laccase on the concentration of organic cosolvents (alcohols, glycols and formamides) in mixed aqueous media has a pronounced threshold character: it does not change up to a critical concentration of the non-aqueous cosolvents added, yet further increase of the latter (by only a small percentage, by vol.) leads to an abrupt decrease in enzyme activity. Fluorescence studies indicate that the inactivation results from reversible conformational changes (denaturation) of the enzymes. There is a linear correlation between the critical concentration of residual water (at which the enzyme inactivation occurs in a threshold manner) and the hydrophobicity of the organic cosolvents added. A quantitative criterion is suggested for the selection of organic cosolvents to be used for enzymatic reactions in homogeneous water/organic solvent media.The behaviour of enzymes in mixed aqueous media has been studied extensively since the fifties (see the first review on this subject by Singer [l]). There are at least two reasons for this. First, such studies help us to clarify the contribution of different molecular forces to maintaining the native structure of the protein [l, 21 and to get a deeper insight into the structure/stability relationships in proteins in general [3]. Second, a number of enzyme-catalysed processes, such as syntheses of peptides and esters, transformation of some hormones, fats and steroids, etc. must be performed in media with a low water content. The reasons for this, e.g. the increase in solubility of poorly water-soluble natural and organic compounds and/or a thermodynamic shift of the chemical equilibrium toward the desired products (and other applied aspects), have been frequently discussed in the literature (for reviews, see [4 -81).A typical experiment designed to elucidate the effect of water-miscible organic solvents on proteins is usually performed as follows: increasing amounts of the organic cosolvents are added to an aqueous solution of the enzyme and the manner is studied in which its structure (as assayed by physical methods) and/or its catalytic activity change. The plots of protein spectral characteristics versus concentrations of an organic cosolvent are rather informative, since they have, as a rule, a pronounced threshold profile (for some examples, see [I, 9, 101). Hence, after a critical concentration of organic cosolvent (20-50% by vol. usually) has been achieved, the spectra1 characteristics of the protein change abruptly. This is strong evidence of conformational rearrangements in the structure of the protein, i.e. of its denaturation.Correspondence to V. V. Mozhaev, Chemistry Department, Abbreviation. C50r the concentration of solvent at which half Enzymes. a-Chymotrypsin

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To simulate in vitro the conditions under which enzymes act in vivo, enzyme molecules have been entrapped in hydrated reverse micelles of a surfactant in organic solvents. In this system the catalytic activity of one of the enzymes studied (peroxidase) became much higher than in water, and the specificity of the other enzyme (alcohol dehydrogenase) was dramatically altered.

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The Phenomenon of the regulation of the catalytic activity of enzymes via changing their oligomeric composition in the system of reversed micelles of sodium bis(2‐ethylhexy)sulfosuccinate (AOT) in octane was studied using z‐chymotrypsin (CT) from bovine brain and alkaline phosphatase (AP) from calf intestinal mucosa. The dependences of the enzyme catalytic activity on the AOT hydration degree (W=[H2O][AOT]), the parameter determining the radius (r 4) of the inner cavity of micelles, usually represent the bell‐shaped curves. The maximal catalytic activity is observed at such Wo when r 4 is equal to the size of the enzyme molecule. The position of this maximum strictly correlates with the enzyme oligomeric composition. Thus, in the case of CT this is observed at Wo =12 when r 1 is equal to the radius (rp ) of the CT globule. In the case of artificially produced conjugate containing six cross‐linked CT molecules, this is observed at W o=43 when r 4 is equal to the radius of the sphere surrounding the absolute octahedron composed of six CT globules. The dependence of the catalytic activity of AP on Wo represent a curve with two maxima that are observed when r 4 is equal to r p of either AP monomer (W=17) or AP dimer (W o=25), Ultracentrifugation experiments revealed that variation of W o causes a change in the oligomeric composition of AP its transition from monomeric (W o<20) to demeric form (W o > 20). Hence, the observed maxima correspond to functioning of different oligomeric forms of AP.

The properties of penicillin acylase from E. co/i solubilizcd by hydrated reversed micdles (RM) of Aerosol OT in octane wcrc studied. The dependence of catalytic activity on the hydration degree, a parameter which determines the size of the micclle inner cavity, has a curve with three optima, each one corresponding to the enzyme functioning either in a dimer form (w, = 23) or i a form of scparatc subunits, a heavy one, fi, and a light one, CL (IV, = 20 and 14, respectively). The reversible dissociation of the enzyme was confirmed by ultracentrifugation followed by electrophoresis.Penicillin acylase; Protein subunit; Micellar enzymology; Reversed micellc 1, INTRODUCTION Penicillin acylase (PA) from ~5. cofi (EC 3.5.1.11) consists of two non-covalently bound subunits (M, 23,000 and 62,000, respectively), which are processed from the common polypeptide precursor [1,2]. The role of such structural organization is not yet clear, but the active site of the enzyme is thought to be located on the heavy /?-subunit, while the light a-subunit is responsible for substrate specificity and participates in binding penicillin side chains [3]. The suggestion that the formation of the active site should involve the association of both subunits has been formally approved by experiments on subunit separation which resulted in the entire loss of enzyme activity [3,4]. Moreover, a fact which should be taken into account is that according to the traditional methods of enzymology the dissociation of oligomeric enzymes (the first step in the separation of subunits) is usually accomplished under strongly denaturing conditions [S], and the above attempts [3,4] were not an exception.A new approach to the analysis of structure-function relationships in oligomeric enzymes, which includes a 'soft' disassembling of subunit complexes in non-denaCorrespondence ah'ress:

Hydrated reversed micelles of Aerosol OT (AOT) in octane have been studied by 13C NMR spectroscopy. The changes of spin-lattice relaxation times (T1) for individual segments of the AOT molecule, induced by entrapping a protein (α-chymotrypsin) into the micelle, have been determined by the inversion-recovery technique. The dramatic (three-fold) increase of T1 found for the α-CH2 groups in the AOT molecules indicates that (unlike in the unfilled micelle) in the protein-containing micelle the boundary of the water cavity is shifted outward (0.5-0.7 nm, under the given experimental conditions), the alkyl chains of the surfactant being “flooded” by water molecules. This observation explains why the outer size of the reversed micelle does not change on insertion of a bulky protein molecule.