Molecular packing tunes the activity of Kluyveromyces lactis beta-galactosidase incorporated in Langmuir-Blodgett films

EDUARDO M. CLOP; PEDRO D. CLOP; JULIETA M. SÁNCHEZ; MARIA A. PERILLO

LANGMUIR

AMER CHEMICAL SOC

Lugar: Washington; Año: 2008 vol. 24 p. 10950 - 10960

0743-7463

Functional consequences of constraining B-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 ß-Gal, were obtained by transferring Langmuir films (L) initially packed at 15 and 35 mN/m, respectively, to alkylated glasses. The B-Gal-monolayer binding equilibrium, mainly the adsorption rate and affinity, depended on the initial monolayer’s surface pressure (lower for higher pi). At pi = 15 and 35 mN/m, the surface excess (G) followed downward parabolic and power-law tendencies, respectively, as a function of subphase protein concentration. G values in L roughly reflected the protein surface density chemically determined in LBs (0-7.5 ng/mm2 at pi = 0-35 mN/m and [B-Gal]subphase = 0-100 µg/mL). The B-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 Hill’s model (K0.5 = 9.55 ± 0.4 mM, Vmax,35 = 0.0021 µmol/min/mg protein, Hill coefficient n = 9) and Savageau’s 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.