The size of the inner water cavity of reversed micelles formed in a triple system 'water-surfactant-organic solvent' can be widely varied by changing the degree of surfactant hydration. This gives grounds to use reversed micelles as matrix microreactors for the design of supramolecular complexes of proteins. Using ultracentrifugation analysis, it has been demonstrated that the oligomeric composition of various enzymes (ketoglutarate dehydrogenase, alkaline phosphatase, lactic dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase) solubilized in reversed micelles of Aerosol OT [sodium bis(2-ethylehexyl)sulfosuccinate] in octane changes upon variation of the degree of hydration. An oligomeric complex forms under conditions when the radius of the micelle inner cavity is big enough to incorporate this complex as a whole. At lower degrees of hydration the micelles 'uncouple' such complexes to their components. The catalytic properties of various oligomeric complexes have been studied. Possibilities of using reversed micelles for the separation of subunits of oligomeric enzymes under non-denaturating conditions have been demonstrated. In particular, the isolated subunits of alkaline phosphatase, lactic dehydrogenase and glyceraldehyde-3-phosphate have been found to be active in Aerosol OT reversed micelles. The dependences of the catalytic activity of oligomeric enzymes represent saw-like curves. The maxima of the catalytic activity observed at these curves relate to the functioning of various oligomeric forms of an enzyme. The radii of the micelle inner cavity under conditions when these maxima are observed correlate with the linear dimensions of the enzyme oligomeric forms. Correlation of the position of a maximum with the shape of an oligomeric complex is discussed.
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:
(1S,4R)-4-Hydroxycyclopent-2-enyl-acetate (1), an attractive starting material for the synthesis of prostaglandins, was readily prepared by an enzyme-catalyzed interesterification procedure using acetic anhydride as acylation agent. As the chemical yield of the chiral monoacylation product is rather low (45%), we investigated the acylation mechanism of this reaction to optimize the product output. Kinetic measurements were carried out by means of gas chromatography on a chiral stationary phase, synthesized by methylation of β-cyclodextrin.
Reversed micelles of a surfactant were formed in ternary systems "water immiscible organic solvent-surfactant-water (buffer)" within determined quantitative relations of the components. They are well defined by size and structure. The degree of hydration of the surfactant (the so-called w,) also dictates the size of the inner cavity of the reversed micelle and thus the physico-chemical characteristics of the water in the inner cavity, which differ from those of bulk water. Enzymes entrapped in the inner cavity of the reversed micelle retain their catalytic activity and their substrate specifity. The dependence of the rate of the enzymatic reaction on the wn was, in the case of monomeric enzymes, a bell-shaped function. As a rule, the optimum catalytic activity was found near degrees of hydration (wo) ensuring by theoretical consideration, an accordance of the enzyme size with the size of the inner cavity of the reversed micelle.The investigation (performed for the first time as far as we know) of the characteristics of an oligomeric enzyme in the system of reversed micelles was carried out using an oligomeric enzyme with identical subunits: lactate dehydrogenase (EC 1.1.1.27), isoform M4 from pig muscle. We have shown that the dependence of the catalytic activity of LDH on w, is characterized by several optima.Based upon these results it seems probable that LDH is able to exist in the system of reversed micelles in different oligomeric forms, each of which is catalytically active. That means, there exist single optimal wn (co-relating with the size of the inner cavity of the reversed micelle) for the catalytic activity of each of the protein aggregates. This hypothesis was confirmed by an independent method: ultracentrifugation. Ultracentrifugation of reversed (Aerosol OT) micelles is a convenient methodology for determining molecular masses of proteins (Khmelnitsky et al., 1982). By this means, and taking into consideration physical data of the LDH described in the literature, we were able to show that at least 85% of the LDH protein exists in the reversed micelle: at Wn = 14 as a monomer, at wn = 18 as a dimer and at 65 Biocatal Biotransformation Downloaded from informahealthcare.com by Freie Universitaet Berlin on 11/19/14
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