Dual substrate/solvent- roles of water and mixed reaction-diffusion control of β-Galactosidase catalyzed reactions in PEG-induced macromolecular crowding conditions

NOLAN, VERÓNICA; CLOP, PEDRO D.; BURGOS, M. INÉS; PERILLO, MARÍA A.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACADEMIC PRESS INC ELSEVIER SCIENCE

Año: 2019 vol. 515 p. 190 - 195

0006-291X

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.