The effect of molecular crowding on β-Galactosidase activity revisited.

NOLAN, MARÍA VERÓNICA; BURGOS, MARTHA INÉS; PERILLO, MARÍA ANGÉLICA

Tucuman

Congreso; . XXXIV Reunión Anual de la Sociedad Argentina de Biofísica; 2016

Sociedad Argentina de Biofísica

Enzymatic activities were historically assayed in dilute solutions where molecular crowding, molecular confinement and their consequences were not taken into account. Here we report how macromolecular crowding tunes catalytic parameters for the tetrameric β-Galactosidase (-Gal) from Escherichia coli. Previously, we reported that molecular crowding modified -Gal enzymatic activity. Using o-nitrophenyl--galactopyranoside (ONPG) as substrate and polyethylene glycol (PEG6000) (1) as crowding agent, we demonstrated that kinetic parameters were affected: Vmax was slightly diminished, while the affinity of the enzyme-substrate interaction (KM) suffered a significant decrease at growing molecular crowding levels. It is known that in the mechanism of the -Gal catalyzed ONPG hydrolysis, water participates in the rate limiting step of the reaction. So, our results suggest that in the presence of PEG, the availability of water as a substrate which is partially lowered with respect to PEG-free media (2) is affecting the reaction. In the present work, we reanalyzed these results under the Eadie-Hofstee model. Another substrate, p-nitrophenyl--galactopyranoside (PNPG), whose reaction mechanism did not involve water in the rate limiting step, was also used to test the effect of the change in water availability on the enzyme activity