The effect of molecular crowding on β-Galactosidase activity revisited

BURGOS I; NOLAN MV; PERILLO MA

Tucumán

Congreso; III LAFeBS- IX IberoAmerican Congress of Biophysics ? SAB2016; 2016

Previously wereported that molecular crowding modified b-Galactosdase (β-Gal) enzymaticactivity. Using o-nitrophenyl-b-galactopyranoside(ONPG) as substrate and polyethylene glycol (1) as crowding agent wedemonstrated that kinetic parameters were affected: Vmax wasslightly diminished, while the affinity of the enzyme-substrate interaction (KM)suffered a significant decrease at growing molecular crowding levels. In thepresent work, we reanalyzed these results under the Eadie-Hofstee model. Wefound a biphasic behavior in the presence of PEG with two components, one ofthem resembling the behavior in dilute media and another one exhibiting an affinity decreasing in a PEGconcentration-dependent manner. It is known that in the mechanism of the β-Gal catalyzed ONPGhydrolysis, water participates inthe rate limiting step of the reaction. So, our results suggest that in thepresence of PEG, the availability of water as a substrate which is partially loweredwith respect to PEG-free media (2) is affecting the reaction. The biphasickinetics of hydrolysis was not observed with the substrate p-nitrophenyl-b-galactopyranoside(PNPG) whose reaction mechanism did not involve water in the rate limitingstep. Similar results were reported for β-Gal entrapped in mesoporous silicate matrix (3). Bothenvironments, the mesoporous matrix and PEG solution have in common thepresence of more than one population of water molecules differing in theirmobility. Thus, taken together our results strongly suggest that restrictions in the water availabitlity emerges as ageneralized phenomenon affecting the rate limiting step of b-Gal catalyzedreaction