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

Functional consequences of constraining beta-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 beta-Gal, were obtained by transferring Langmuir films (L) initially packed at 15 and 35 mN/m, respectively, to alkylated glasses. The beta-Gal-monolayer binding equilibrium, mainly the adsorption rate and affinity, depended on the initial monolayer's surface pressure (lower for higher pi(i)). At pi(i) = 15 and 35 mN/m, the surface excess (Gamma) followed downward parabolic and power-law tendencies, respectively, as a function of subphase protein concentration. Gamma values in L roughly reflected the protein surface density chemically determined in LBs (0-7.5 ng/mm(2) at pi(i) = 0-35 mN/m and [beta-Gal](subphas), = 0-100 mu g/mL). The beta-Gal-catalyzed hydrolysis of o-nitrophenyl-galactopyranoside showed a Michaelian kinetics in solution as well as in LB15, K-M, K-M,K-LB15, V-max, and V-max,V-LB15 were 5.15 +/- 2.2 and 9.25 +/- 6 mM and 39.63 and 0.0096 +/- 0.0027 mu mol/min/mg protein, respectively. The sigmoidal kinetics observed with LB35 was evaluated by Hill's model (K-0.5 = 9.55 +/- 0.4 mM, V-max,V-35 = 0.0021 mu mol/min/mg protein, Hill coefficient n = 9) and Savageau's fractal model (fractal constant K-f = 9.84 mM; reaction order for the substrate g(s) = 9.06 and for the enzyme g(e) = 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.