Enzyme catalysis in water pools

Menger, Fredric M.; Yamada, Kohei

doi:10.1021/ja00516a039

Cited by 274 publications

(131 citation statements)

References 2 publications

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“…6) is slightly higher than that observed in aqueous solution (broken line) [151,216,2471. More significant 'superactivity' was found for peroxidase [150,182,219,2531.…”

Section: Effects Of Water Contentmentioning

confidence: 70%

Micellar enzymology

Martínek

Levashov

Klyachko

et al. 1986

European Journal of Biochemistry

547

248

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Experimental approaches to modelling the enzymatic function of biological membranes are discussed. Emphasis is given to pseudohomogeneous systems such as proteolipid complexes and enzymes in organic solvents; the latter are solubilized with phospholipids or synthetic surfactants. Methods for producing and studying such micellar systems are considered. The key research problems of micellar enzymology are formulated and its relation to enzyme membranology is discussed. Finally, the new potentialities are noted of applied enzymology (biotechnology) offered by application of a colloidal solution of water in organic solvents as a microheterogeneous medium for enzymatic reactions.Until now the development of molecular enzymology has been mainly directed to studying free enzymes [l -41. In other words, the most valuable experiments aimed at elucidating the structure of catalytic centers and the physico-chemical mechanisms of biocatalysis were successful only with the enzymes isolated from the living cells in quite a pure form. However, such 'pure' experiments, as was noted time and again [5], naturally raise the question as to whether the enzyme properties observed in vitro can be correlated adequately with the conditions of its functioning in vivo. Such a doubt is quite equitable since it became clear [6 -91 that the subcellular structure and the compartmentalization of enzymes play the most important role in metabolism regulation.Thus, the essence of the contradiction is that in vitro studies of enzymes are usually conducted in water (buffer). However, in the living cell, enzymes mostly act on or near the 'water/ organic medium' interface. For instance, many enzymes, if not the majority, are located on the surface of biological membranes or inside them [lo-161. Other enzymes function in mobile complexes with macromolecular components of the cell, e.g. with proteins or polysaccharides [14 -161. Generally speaking, the boundary between 'bound' and 'diffusion-free' enzymes is rather conventional, and such a classification is more methodical (depending on the ease of the enzyme isolation) than functional (the enzyme localization in the cell) [16]. The problem is that many of the enzymes bind to the membrane surface loosely and the degree of their binding depends on the concentration of metabolites [14-191. The physiological state of the cell determines not only the adsorption of some enzymes but their possible translocation through membranes [20,21]. In the adsorbed state in vivo (on the enterocyte brushborder) extracellular enzymes were found as well, such as proteases of the digestive tract, namely trypsin and a-chymotrypsin [22, 231, which up to now have remained model subjects of the classical 'in-water' enzymology.Finally, a few words about the medium in which diffusionfree enzymes function in the cell (for a review see [24]). It is not very clear why macromolecules (immunoglobulins and albumin) injected into the living cell diffuse so slowly (as in a 60% sucrose solution) [25]. The comparison of the diffusion rat...

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“…6) is slightly higher than that observed in aqueous solution (broken line) [151,216,2471. More significant 'superactivity' was found for peroxidase [150,182,219,2531.…”

Section: Effects Of Water Contentmentioning

confidence: 70%

Micellar enzymology

Martínek

Levashov

Klyachko

et al. 1986

European Journal of Biochemistry

547

248

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“…Dependence of k,,, and K, on w, Since the first studies on enzymes solubilized in reverse micelles, it is known that w, is an important parameter which strongly affects the catalytic activity of the solubilized enzyme [20,21]. The most common dependence of enzyme activity on w, is bell-shaped.…”

Section: Inhibition By Product In Reverse Micellesmentioning

confidence: 99%

Product inhibition of α‐chymotrypsin in reverse micelles

Bru

Walde

1991

European Journal of Biochemistry

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The a-cliymotrypsin-catalyzed hydrolysis of succinyl-~-alanyl-~-alanyl-~-prolyl-~-phenylalanyl p-nitroanilide has been studied in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. It has been found that a-chymotrypsin is strongly inhibited competitively by the acidic peptide product which is formed during the course of the reaction. It has also been shown that the application of the integrated form of the Michaelis-Menten equation can be useful to detect possible inhibition effects and abnormal kinetic behavior of enzymes in reverse micelles. Furthermore, it has been shown that the turnover number (k,,,) at low water content is lower than in water and increases as the water content in the system (w, = One of the most surprising findings for (certain) enzymes in reverse micelles is 'superactivity', a term which is currently being used in the literature on micellar enzymology for describing the fact that the turnover number, k,,,, is, for some enzyme/substrate systems in reverse micelles, higher than in water [16, 191. Different research groups have reported that a-chymotrypsin belongs to the 'superactive' enzymes [20 -231. Although a-chymotrypsin is the most intensively studied enzyme in reverse micelles, discrepancies can be found in the literature, mainly those concerning the dependence of the enzyme activity on w, [21-231. The present paper should help in understanding the reason for some of these discrepancies. Throughout this paper we will consider in more detail the kinetics of the a-chymotrypsin-catalyzed hydrolysis of succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl p-nitroanilide (abbreviated as Suc-Ala-Ala-Pro-Phe-NH-Np) in reverse micelles formed by AOT [sodium bis(2-ethylhexyl)sulfosuccinate] in isooctane.We will first put forward experimental evidence which shows that the kinetic behavior of a-chymotrypsin against the chromogenic tetrapeptide Suc-Ala-Ala-Pro-Phe-NH-Np resembles that in water as w, is increased (i.e. as the reverse micelles are enlarged).Secondly, we will demonstrate that a-chymotrypsin is strongly inhibited in reverse micelles by product, the overall inhibition constant being up to almost 400 times lower than in bulk water.Special attention has to be paid to the substrate, Suc-AlaAla-Pro-Phe-NH-Np, since it is one of the 'best' synthetic substrate for a-chymotrypsin: both high affinity (i.e. low K, value) and high reactivity (i.e. high k,,,) have been reported [24]. Thus, analysis of the time course of the reaction in reverse micelles can easily be performed by using the integrated Michaelis-Menten equation which has never been used before for analyzing enzyme-catalyzed reactions in reverse micellar systems. MATERIALS AND METHODS ReagentsAOT was a product from Sigma (USA) and was used as obtained. Newer batches of Sigma AOT are of satisfactory

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“…We use the purification method published elsewhere [35] and the surfactant purity was evaluated using gas chromatography [36]. The polymer poly(maleic anhydride-alt-1-octadecen), PMAO (M r = 40 kDa) was from Sigma-Aldrich and was used as received without further purification.…”

Section: Reagents and Vesicle Preparationmentioning

confidence: 99%

Spontaneous Vesicles Modulated by Polymers

2011

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Abstract:Vesicles are widely used in technological applications including cosmetic products, in microencapsulation for drug delivery, as anticancer agents and in the technology of adhesives, paints and inks. The vesicle size and the surface charge are very important properties from a technological point of view. Thus, the challenge in formulation is to find inexpensive stable vesicles with well-defined sizes and to modulate the surface charge of these aggregates. In this work we analyze the effect of different polymers on the structural properties of vesicles of the biodegradable surfactant sodium bis(2-ethyl-hexyl) sulfosuccinate, Aerosol OT. Using fluorescence, conductivity, electrophoretic mobility and dynamic light scattering measurements we study the effect of the polymer nature, molecular weight and polymer concentration on the stability and the vesicle size properties. Results demonstrate that it is possible to modulate both the size and the electric surface charge of spontaneous vesicles of Aerosol OT by the addition of very small percentages of poly(allylamine) and poly(maleic anhydride-alt-1-octadecen).

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Enzyme catalysis in water pools

Cited by 274 publications

References 2 publications

Micellar enzymology

Micellar enzymology

Product inhibition of α‐chymotrypsin in reverse micelles

Spontaneous Vesicles Modulated by Polymers

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