Enzyme kinetics in reversed micelles

Abstract

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...

Enzyme kinetics in reversed micelles

“…In all cases, values of k,, around lo6 M-' s-' gave a good simulation. These values for k,, agree well with literature data, but are lower than the exchange rates reported previously [19]. An increase in k,, enhanced the calculated rate at low NADH concentrations, giving a poorer correlation.…”

“…From the concentrations of reversed micelles and the volume fraction of water given in Table 1, the intramicellar NADH concentration in AOT-reversed micelles can be calculated to be 75 mM, much higher than the values in CTAB-reversed (27 mM) and Triton-reversed micelles (37 mM). Similar results have been obtained for enoate reductase [19]. Figs 6A and 7 A show the plots for CTAB-and Triton-reversed micelles at high ionic strength, using the kinetic parameters of the enzyme measured in aqueous solution at high ionic strength.…”

“…In the case of reversed micelles, the charge of the head groups might also play a role [9,10,19,201, as has been observed for immobilized enzymes 121 -241. We investigated the effect on the kinetic parameters of the enclosure of 208-hydroxysteroid dehydrogenase in reversed micelles composed of a cationic, an anionic or a nonionic surfactant.…”

“…The basic thought behind this theory is that V, K A and KH for 20P-hydroxysteroid dehydrogenase in reversed micelles have the same values as in an aqueous solution, and that deviations from the behaviour in aqueous solutions are caused by confinement of the enzyme to the water pool of reversed micelles. This approach has been shown to be valuable in explaining the behaviour of enoate reductase in reversed micelles [19].…”

“…Values for the parameters used are listed in Table 1. Concentrations of AOT-reversed micelles [ 161, concentration of CTAB-reversed micelles [17], k,, for CTAB [18] and concentration of Triton-reversed the micelles [19] were derived from references. k,, and kin were adjusted to give the best fits, k,,, and k,, are related by the partition coefficient using the formula [l 11:…”