Universidad Nacional de Córdoba, Facultad de Ciencias Exactas, Físicas y Naturales. ICTA and Departamento de Química, Cátedra de Química Biológica, Av. Vélez Sársfield 1611, 5016, Córdoba, Argentina; CONICET-Universidad Nacional de Córdoba, Instituto de Investigaciones Biológicas y Tecnológicas (IIByT). Córdoba, Argentina. Electronic address: [Email]
Here we studied the effect of molecular crowding on the hydrolysis of ortho- and para-nitrophenyl-β-D-galactopyranosides (ONPG, PNPG) catalysed by Escherichia coli β-Galactosidase in the presence of 0-35%w/v 6kD polyethyleneglycol (PEG6000). The Eadie-Hofstee data analysis exhibited single straight lines for PNPG at all [PEG6000] as well as for ONPG in the absence of PEG6000 so a Michaelian model was applied to calculate the kinetic parameters KM and kcat (catalytic rate constant) values. However, for ONPG hydrolysis in the presence of PEG6000, the two slopes visualized in Eadie-Hofstee plots leaded to apply a biphasic kinetic model to fit initial rate vs. [ONPG] plots hence calculating two apparent KM and two kcat values. Since the rate limiting-step of the enzymatic hydrolysis mechanism of ONPG, but not of PNPG, is the water-dependent one, the existence of several molecular water populations differing in their energy and/or their availability as reactants may explain the biphasic kinetics in the presence of PEG6000. With PNPG, KM as well as kcat varied with [PEG6000] like a parabola opening upward with a minimum at 15 %w/v [PEG6000]. In the case of ONPG, one of the components became constant while the other component exhibited a slight increasing tendency in kcat plus high and [PEG6000]-dependent increasing KM values. Sedimentation velocity analysis demonstrated that PEG6000 impaired the diffusion of β-Gal but not that of substrates. In conjunction, kinetic data reflected complex combinations of PEG6000-induced effects on enzyme structure, water structure, thermodynamic activities of all the chemical species participating in the reaction and protein diffusion.