Cationic Reverse Micelles Create Water with Super Hydrogen‐Bond‐Donor Capacity for Enzymatic Catalysis: Hydrolysis of 2‐Naphthyl Acetate by α‐Chymotrypsin

Abstract

Reverse micelles (RMs) are very good nanoreactors because they can create a unique microenvironment for carrying out a variety of chemical and biochemical reactions. The aim of the present work is to determine the influence of different RM interfaces on the hydrolysis of 2-naphthyl acetate (2-NA) by alpha-chymotrypsin (alpha-CT). The reaction was studied in water/benzyl-n-hexadecyldimethylammonium chloride (BHDC)/benzene RMs and, its efficiency compared with that observed in pure water and in sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT) RMs. Thus, the hydrolysis rates of 2-NA catalyzed by alpha-CT were determined by spectroscopic measurements. In addition, the method used allows the joint evaluation of the substrate partition constant K(p) between the organic and the micellar pseudophase and the kinetic parameters: catalytic rate constant k(cat), and the Michaelis constant K(M) of the enzymatic reaction. The effect of the surfactant concentration on the kinetics parameters was determined at constant W(0)=[H(2)O]/[surfactant], and the variation of W(0) with surfactant constant concentration was investigated. The results show that the classical Michaelis-Menten mechanism is valid for alpha-CT in all of the RMs systems studied and that the reaction takes place at both RM interfaces. Moreover, the catalytic efficiency values k(cat)/K(M) obtained in the RMs systems are higher than that reported in water. Furthermore, there is a remarkable increase in alpha-CT efficiency in the cationic RMs in comparison with the anionic system, presumably due to the unique water properties found in these confined media. The results show that in cationic RMs the hydrogen-bond donor capacity of water is enhanced due to its interaction with the cationic interface. Hence, entrapped water can be converted into "super-water" for the enzymatic reaction studied in this work.

Interfacial water with special electron donor properties: Effect of water–surfactant interaction in confined reversed micellar environments and its influence on the coordination chemistry of a copper complex

“…In this way, recently we have shown that the interfacial water properties can be very different depending on the charge of the surfactant polar head group [65,66]. Specifically, we have found different behavior of the fluorescent cationic hemicyanine trans-4-[4-(dimethylamino)-styryl]-N-methylpyridinium iodide, depending on whether the probe is solvated by the water in AOT or in BHDC reversed micellar systems.…”

“…The results suggested that the water molecules sequestrated inside AOT and BHDC reversed micellar systems show important differences in its properties because they interact in different form with the surfactants polar head [65]. Also, quite recently, we have investigated the influence of different reversed micellar interfaces on the hydrolysis of 2-naphthyl acetate by achymotrypsin [66]. This reaction was studied in benzene/BHDC/ water RMs, and its efficiency was compared with that observed in pure water and in AOT RMs.…”

“…This interaction makes the interfacial water much more a hydrogen bond donor than the bulk water. An opposite situation was observed for AOT RMs where the hydrogen bond donor ability is diminished because the water molecules interact strongly with the sulfonate head group by hydrogen bonds [66].…”

Inhibited Phenol Ionization in Reverse Micelles: Confinement Effect at the Nanometer Scale

“…In particular, the water at the cationic RMs interfaces seems to have enhanced hydrogen-bonddonor capability. [27,28,38] Phenols have been used as molecular probes to determine binding constants at the AOT RMs interface [39,40] or to measure pK a values upon confinement. [41][42][43] The behavior of different phenols in RMs were investigated by Magid et al [40] They calculated the binding constants for a series of phenols to AOT RMs and found that the binding process depended mainly on the hydrogen-bonding ability of the phenols to form hydrogen bonds with the AOT polar head groups.…”

“…Also, very recently we found that 2-naphthol converted into 2-naphtholate in BHDC RMs, but not in AOT RMs. [28] We explained this fact by considering differences in the interfacial water properties. Water molecules trapped inside the AOT RMs had enhanced electron-donor abilities than those of bulk water.…”