Activity of Kluyveromyces lactis b-Galactosidase confined in a Langmuir-Blodgett Films

PEDRO D. CLOP; EDUARDO M. CLOP; MARÍA A. PERILLO

Búzios, Rio de Janeiro, Brasil.

Congreso; VII Iberoamerican Congress of Biophysics.; 2009

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In previous studies on b-galactosidase (b-Gal) activity in heterogeneous systems we demonstrated that  not only the soluble enzyme but also the product of the reaction it catalyzes could partition within the interface lipid-water. The ONPG (ortho-nitrophenylgalactopyranoside) (S) hydrolysis catalyzed by b-Gal turned into a cooperative phenomenon when this enzyme was in a bi-dimensional phospholipid film packed at lateral surface pressures Pi ≥35 mN/m (Langmuir-Blodgett (LB) film), prepared by the transference of Langmuir films (L) from the air-water interface to a hydrophobic solid surface. In the present work we measured reaction rates at different enzyme concentrations ([E]). Kinetic data plotted in log-log plots let estimate the kinetic order with respect to S (gs=9.06) and the fractal Michaelis constant (Kf) which resulted an [E]-dependent value. A secondary plot (gs*Log(Kf) vs Log[E]) let calculate the kinetic order with respect to enzyme, resulting a value ge=2.31 close to the fractal dimension of proteins surface. The value [3-ge]=0.69, representing the dimension of the actual space where the reaction takes place, was similar to gs. Fractal reaction orders would reflect the fractal organization of the environment, demonstrated by AFMimages, more than the molecularity of the reaction.