PEG-induced molecular crowding leads to a relaxed conformation, higher thermal stability and lower catalytic efficiency of Escherichia coli β-galactosidase.

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 from Escherichia coli, β-Gal. We detected increases in KM (weaker substrate binding) and a nonlinear variation in Vmax, with a minimum at 25% W/P of the crowding agent (polyethyleneglycol molecular mass 6000, PEG(6000)) resulting in a linear decrease in the catalytic efficiency (kcat/KM) within the whole [PEG(6000)] range tested). Presence of crowding agent affected β-Gal structural content and increased its thermal resistance. Steady state fluorescence and Fourier transformed infrared spectroscopic observations are compatible with crowding-induced disordering and restricted internal dynamics as a result of excluded volume and solvent structuring effects. This leads to a non-optimal substrate-binding site and a less conformationally strained protein.