INVESTIGADORES
CLOP Eduardo Matias
artículos
Título:
Molecular Packing Tunes the Activity of Kluyveromyces lactis b-Galactosidase Incorporated in Langmuir-Blodgett Films
Autor/es:
CLOP, E. M.; CLOP, P. D; SANCHEZ, J. M.; PERILLO, M.A.
Revista:
LANGMUIR
Editorial:
American Chemical Society
Referencias:
Lugar: New York, USA; Año: 2008 vol. 24 p. 10950 - 10960
ISSN:
0743-7463
Resumen:
Functional consequences of constraining β-Gal in bidimensional space were studied at defined molecular packing densities and constant topology. Langmuir−Blodgett films, LB15 and LB35 composed of dipalmitoyl phosphatidylcholine and K. lactis β-Gal, were obtained by transferring Langmuir films (L) initially packed at 15 and 35 mN/m, respectively, to alkylated glasses. The β-Gal-monolayer binding equilibrium, mainly the adsorption rate and affinity, depended on the initial monolayers surface pressure (lower for higher πi). At πi = 15 and 35 mN/m, the surface excess (Γ) followed downward parabolic and power-law tendencies, respectively, as a function of subphase protein concentration. Γ values in L roughly reflected the protein surface density chemically determined in LBs (0−7.5 ng/mm2 at πi = 0−35 mN/m and [β-Gal]subphase = 0−100 μg/mL). The β-Gal-catalyzed hydrolysis of o-nitrophenyl-galactopyranoside showed a Michaelian kinetics in solution as well as in LB15. KM, KM,LB15, Vmax, and Vmax,LB15 were 5.15 ± 2.2 and 9.25 ± 6 mM and 39.63 and 0.0096 ± 0.0027 μmol/min/mg protein, respectively. The sigmoidal kinetics observed with LB35 was evaluated by Hills model (K0.5 = 9.55 ± 0.4 mM, Vmax,35 = 0.0021 μmol/min/mg protein, Hill coefficient n = 9) and Savageaus fractal model (fractal constant Kf = 9.84 mM; reaction order for the substrate gs = 9.06 and for the enzyme ge = 0.62). Fractal reaction orders would reflect the fractal organization of the environment, demonstrated by AFM images, more than the molecularity of the reaction. Particular dynamics of the protein−lipid structural coupling in each molecular packing condition would have led to the different kinetic responses.