Water state/structure inside nanopores modulates hydrolytic enzymatic catalysis

BURGOS, M. INES; AYLÉN OCHOA; MANUEL VELASCO; MARÍA ANGÉLICA PERILLO; RODOLFO ACOSTA

San Luis

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Sociedad Argentina de Biofísica

It is widely accepted that water molecules inside the cell compartments exhibit physical properties that differ of dilute solutions. Then, the microenvironment in which metabolic activity actually occurs in cells is not equivalent to the conditions used to determine the properties of enzymes in vitro. In this work the kinetic activity of β-Galactosidase confined in a nanopore of a silicate gel was studied and it was related to confined water dynamics. Encapsulation of proteins in silicate gels obtained by the sol-gel method has been of interest for the possibility of studying protein properties in conditions far fromdilute solutions. The silicate gel obtained showed highly porous structure, quite narrow pore diameter distribution, around 40 nm, and high surface area. The results observed for the encapsulated β-Galactosidase (Eβ-Gal), which catalyses a hydrolysis reaction, let us take into consideration the differences between bulk and confined water moleculesinside the nanopores and the influence of these differences on the catalytic activity. The activity of Eβ-Gal was measured for two substrates: PNPG and ONPG. It is well known from mechanism studies that, for ONPG, the rate limiting step is the one in which a molecule of water takes part in the reaction, while for PNPG the rate limiting step is the previous one in which water is not relevant. The Vmax value calculated, after fitting with Michaelis- Menten model, for Eβ-Gal was higher than the Vmax obtained for the free soluble enzyme (Sβ-Gal) when the substrate hydrolysed was ONPG, and was even higher for the enzymeencapsulated in aged gels. On the other hand, the Vmax values for Eβ-Gal calculated from the measurements with PNPG did not evidence significant differences with Sβ-Gal. Vmax is proportional to the catalytic constant (kc) which, in turn, measures the rate of the limiting step in the reaction mechanism. Strikingly, transversal relaxation times (T2) from 1H-NMR experiments of the hydrogens of water molecules inside the nanopores of the gel, evidenced three population groups and, in all cases, with lower diffusion rate thanthe bulk water molecules. Accordingly, to the results of Vmax with ONPG, the diffusion of the three population of water lowered in aged gels. In view of these results, it is quite plausible to propose that some characteristic of confined water inside the nanopores influence the hydrolytic activity of Eβ-Gal.