Compartamentalization and supramolecular organization as modulatory mechanisms of ƒÒ-galactosidase activity.

SANCHEZ J.M.; TURINA A.DEL V.; CICLIK I.; PERILLO M.A.

Puerto Iguazú- Misiones, Argentina

Congreso; ISN/ASN sponsored satellite meeting; 2001

Heterogeneity is present in natural as well as in artificial systems composed of amphipathic molecules due to their spontaneous tendency to self-aggregate in aqueous media. It is expected that enzyme kinetics would be modulated in those media by three major mechanisms: a) substrate organization, b) partitioning of reactants, enzyme and reaction products between the coexisting phases and c) changes in the intrinsic reaction mechanism due to dimensionality restrictions to diffusion of chemical species (like in a surface) and to modifications of the activation energy barriers. In addition, all those phenomena will be coupled to the supramolecular dynamics of the self-aggregating structures. In the present work we investigated the effect of mechanisms b and c on the activity of  E.coli b-galactosidase towards the artificial and soluble sustrate o-nitro phenyl galactopyranoside (ONPG). Multilamellar vesicles composed of phospholipids having  different degree of saturation in their fatty acid chains, in mixtures with varying proportion of cholesterol or detergents, were used as bilayer membrane models. The effect of pure detergents in the micellar form was also tested. Kinetic parameters, Vmax and KM, determined in the presence and in the absence of bilayers or micelles, showed their clear dependence on several membrane general properties like molecular packing, surface hydration, curvature (measured by quasi-elastic light scattering) and membrane topology. Beta-galactosidase activity was modulated according to the enzyme possibility to penetrate or just be adsorbed to a dimensionality restricted space. Enzyme-membrane interaction was confirm by its penetration into monomolecular layers at the air-water interface with a cut-off surface pressure of 30 mN/m. Membrane-water partition coefficients of b-galactosidase, ONPG and ONP determined according to the theory and methodology developed in our laboratory (Perillo & Arce, 1991, J.Neurosci.Meth.36:203), resulted 118, 0 and 51 respectively. A theoretical model of two-compartments (membrane and aqueous phases), with freely accessible substrate and a nucleophile-like activatory effect on the reaction kinetics exerted at the membrane compartment and possibly due to the structured condition of water molecules, reproduced the lipid concentration dependence we had observed in  Vmax  values. Enzyme but not ONP partitioning towards the membrane was the determinant factor of that behavior. The present may be a good model to understand the modulation of gangliosides digestion which is known to take place after their endocytosis as components of intraendosomal and intralysosomal vesicles, by water-soluble lysosomal acid exohydrolases.