Description of enzyme kinetics in reversed micelles

Verhaert, R.M.D.; Hilhorst, Riet; Vermuë, M.H.; Schaafsma, T.J.; Veeger, C.

doi:10.1111/j.1432-1033.1990.tb15277.x

Cited by 90 publications

(56 citation statements)

References 27 publications

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“…Recently, such partitioning of solubilizates inside reverse micelles has also been taken into account in different theoretical treatments of enzyme kinetics in reverse micelles [.SO-521. It has also been reported in the literature [51] that inhibition effects caused by substrate start to occur when the substrate concentration is of the same order of magnitude as the concentration of reverse micelles. We have not investigated whether these findings are also true in the case of the product inhibition reported here.…”

Section: Application Of the Integrated Michaelis-menten Equation : Sumentioning

confidence: 82%

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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Section: Application Of the Integrated Michaelis-menten Equation : Sumentioning

confidence: 82%

Product inhibition of α‐chymotrypsin in reverse micelles

Bru

Walde

1991

European Journal of Biochemistry

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“…There are several theories in the literature that attempt to correlate trends in enzyme activity with w o to enzyme c~nformation,~ localized substrate and charge concentration profiles,16 and intermicellar exchange rates. 21 We do not speculate on the rationale for the observed trend and simply state that eficient operation can be achieved at wo values above 5. reaction. The data thus appear to validate a stepwise polymerization mechanism where each coupling of a growing chain molecule to a monomer molecule requires a molecule Finally, Figure 11 illustrates the FTIR spectra of the micellar system prior to reaction (case a ) and subsequent to reaction (case b).…”

Section: Effect Of Low Surfactant (Aot) Concentrations On Polyethyl-mentioning

confidence: 94%

Catalytic and interfacial aspects of enzymatic polymer synthesis in reversed micellar systems

Rao

John

Gonzalez

et al. 1993

Biotech & Bioengineering

105

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The enzyme horseradish peroxidase, when encapsulated in reversed micelles, is capable of catalyzing the synthesis of phenolic and aromatic amine polymers. The synthesis of polyethylphenol is specifically considered in this article and is found to be extremely feasible in the micellar system. Polymer chain growth can be controlled to some degree by manipulating the ability of the solvent to sustain chain solubility; this is effectively done by adjusting the surfactant concentration. This results in a degree of control of polymer molecular weight. The synthesized polymer drops out of solution and can be easily recovered.

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“…The obvious conclusion could be that not only apparent K, values but all kinetic parameters in reversed micelles differ from those in aqueous solution. As an alternative, we analyzed the data using the kinetic model proposed in [9]. In this model a distinction is made between the reactions occurring between various reversed micelles and the reactions that can take place inside one reversed micelle (Fig.…”

Section: A 0 T-reversed Mirellesmentioning

confidence: 99%

“…According to this model the rate of the reaction can be described by [9] : The rate of intermicellar exchange of AOT-reversed micelles (kCJ is reported to be in the order of 5 x lo7 (M-' s-') [19]. It has to be stressed that all concentrations are expressed with respect to the total volume of the medium.…”

Section: A 0 T-reversed Mirellesmentioning

confidence: 99%

Enzyme kinetics in reversed micelles

Verhaert

Tyrakowska

Hilhorst

et al. 1990

European Journal of Biochemistry

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Enoate reductase (EC 1.3.1.31) can stereospecificially reduce a variety of a$-unsaturated carboxylates. Its use was extended to apolar media by incorporating the enzyme into a reversed micellar medium. The kinetics of the enzyme in such a medium have been investigated using 2-methylbutenoic acid as substrate and NADH as a cofactor and compared with the reaction rates in aqueous solution.In aqueous solution the enzyme obeys a ping pong mechanism [Buhler et al. (1982) Hoppe-Seyler's Z. Physiol.Chern 363, 609-6251. In 50 mM Hepes pH = 7.0 with ionic strength of 0.05 M the Michaelis constants for NADH and 2-methylbutenoic acid are 20 pM and 6.0 mM respectively. In reversed micelles the kinetics of the reaction (Michaelis constant, maximum velocity as well as inhibitory effects) were markedly different. The rate of the enzymatic reaction of enoate reductase was studied using various concentrations of 2-methylbutenoic acid and various NADH concentrations. In reversed micelles composed of the anionic detergent sodium di(ethylhexyl)sulphosuccinate, the enzymatic reaction deviates substantially from the values in aqueous solution. Using our model (see preceding paper in this issue of the journal), all kinetics could be explained as evolving from enclosure in reversed micelles without any change in the intrinsic rate parameters of the enzyme. So the enzyme itself is unaffected by incorporation in reversed micelles, but the rate of intermicellar exchange as well as the microheterogeneity of the medium, resulting in very high local concentrations of the substrate, are the most important factors altering the reaction pattern. The effect of the composition of the reversed micellar medium was also investigated using either a nonionic or a cationic surfactant. In these solutions too, exchange and microheterogeneity of the medium proved to be the most important parameters influencing the enzymatic reaction. In all reversed micellar solutions inhibition by the enoate was observed at an overall concentration of 0.5-5 mM, implying that a concentration of substrate equal to the K, value in aqueous solution may already cause inhibition in reversed micelles. At this level no inhibition by NADH was observed. The microheterogeneity of the medium also explains this inhibition of the enzyme at relatively low 2-methylbutenoic acid concentrations. [E,], total enzyme concentration expressed with respect to the overall volume; Ka, Michaelis constant for substrate A (NADH); KB, Michaelis constant for substrate B (2-methylbutenoic acid); K",p, apparent Michaelis constant (expressed with respect to the total volume) derived from double-reciprocal plots of l / u versus l/[substrate]; k,,, exchange rate between reversed micellar droplets ( M -' s -'); [MI, concentration of reversed micelles; P, Q, products of the enoate reductase reaction (NAD' and 2-methylbutanoic acid, respectively); u, velocity of the enzyme reaction (s-I); urnax, experimentally determined maximum reaction rate (s-'); V, (intrinsic) rate constant of the forward reaction (M s-I...

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Description of enzyme kinetics in reversed micelles

Cited by 90 publications

References 27 publications

Product inhibition of α‐chymotrypsin in reverse micelles

Product inhibition of α‐chymotrypsin in reverse micelles

Catalytic and interfacial aspects of enzymatic polymer synthesis in reversed micellar systems

Enzyme kinetics in reversed micelles

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