Effect of urea on the enzymatic activity of a lipase entrapped in AOT-heptane-water reverse micellar solutions.
A study has been made of the effect of urea upon the hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by lipase from Rhizopus arrhizus in AOT-heptane-water reverse micellar solutions at pH 7. The partition constants, K, of 2-NA between n-heptane and aqueous urea solutions in the absence of micelles were also determined. It was found that K decreases when the concentration of urea increases. In aqueous solution the rate of hydrolysis of 2-NA catalyzed by lipase is dependent on the concentration of urea (at a given 2-NA concentration). This result can be due to a decrease in the magnitude of the association of lipase with 2-NA and/or to changes in the reaction rate of the lipase-2-NA complex. The modifications of the enzymatic activities elicited by addition of urea show a lineal correlation with K, emphasizing the relevance of hydrophobic effects in the loss of activity. Nevertheless, the slope of the line is higher than one, suggesting that changes in the conformation of the enzyme would be also important. Addition of urea to the micellar solutions provokes a decrease of the enzyme activity. From the dependence of the reaction rate with AOT concentration, the partition constant of 2-NA between n-heptane and the micelles, K(p), was obtained. In the presence of 2 M urea a value of K(p)=0.33 M(-1) was derived. This value is lower than that measured in the absence of urea (Aguilar et al., Arch. Biochem. Biophys. 388 (2001) 231), indicating that incorporation of urea to the micellar interface produces a decrease of the association of 2-NA with the micelles. From a comparison of the results obtained in the micellar solution and in aqueous solution, it is concluded that the enzyme is more resistant to denaturation by urea in the micellar solution than in aqueous solution. Furthermore, at intermediate urea concentrations (2 M), the additive produces an increase in the Michaelis constant (K(M)) without a significant decrease (or even a small increase) in the catalytic rate constant (k(cat)).
Abuin
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Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Correo 33, Santiago, Chile. eabuin@lauca.usach.cl
Lissi
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Solar
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D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D006536:Heptanes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000737:chemistry*; D008823:Micelles; D008856:Microscopy, Fluorescence; D009280:Naphthaleneacetic Acids / Q000737:chemistry; D013997:Time Factors; D014508:Urea / Q000737:chemistry*; D014867:Water / Q000737:chemistry
D006536:Heptanes; D008823:Micelles; D009280:Naphthaleneacetic Acids; D014867:Water; C020424:2-naphthylacetic acid; D014508:Urea; D008049:Lipase