The effect of substrate partitioning on the kinetics of enzymes acting in reverse micelles.
A theoretical model for the expression of enzymic activity in reverse micelles previously developed [Bru. Sánchez-Ferrer & García-Carmona (1989) Biochem. J. 259, 355-361] was extended in the present work. The substrate concentration in each reverse-micelle phase (free water, bound water and surfactant apolar tails) and the organic solvent was expressed as a function of the total substrate concentration, taking into account its partition coefficients, that is, partitioning of the substrate in a multiphasic system. In each phase the enzyme expresses a catalytic constant and a Km. Thus the whole reaction rate is the addition of the particular rates expressed in each domain. This model was compared with that developed for a biphasic system [Levashov, Klyachko, Pantin, Khmelnitski & Martinek (1980) Bioorg. Khim. 6, 929-943] by fitting the experimental results obtained with mushroom tyrosinase (working on both 4-t-butylcatechol and 4-methylcatechol) to the two models. The parameters which characterize reverse micelles, omega 0 (water/surfactant molar ratio) and theta (fraction of water) were investigated. The omega 0 profile was shown to be hyperbolic for both substrates. Activity towards 4-t-butylcatechol decreases as theta increases, this observation being attributable to a dilution of the substrate. A Km of 7.8 M for 4-t-butylcatechol could be calculated on the basis of the biphasic model, whereas it was 13.5 mM when calculating on the basis of our model. A new parameter, rho (= [substrate]/theta), was defined to characterize those substrates that mainly solubilize in the reverse micelle ('micellar substrates').
Bru
R
R
Departamento de Bioquimica, Facultad de Biologia, Universidad de Murcia, Spain.
Sánchez-Ferrer
A
A
García-Carmona
F
F
D001487:Basidiomycota / Q000201:enzymology; D003102:Colloids; D004798:Enzymes / Q000378:metabolism*; D007700:Kinetics; D008823:Micelles; D008954:Models, Biological; D014442:Monophenol Monooxygenase / Q000378:metabolism; D012044:Regression Analysis; D012997:Solvents
D003102:Colloids; D004798:Enzymes; D008823:Micelles; D012997:Solvents; D014442:Monophenol Monooxygenase