Biochemical and Biophysical Research Communications, Vol.515, No.1, 190-195, 2019
Dual substrate/solvent- roles of water and mixed reaction-diffusion control of beta-Galactosidase catalyzed reactions in PEG-induced macromolecular crowding conditions
Here we studied the effect of molecular crowding on the hydrolysis of ortho-and para-nitrophenyl-beta-D-galactopyranosides (ONPG, PNPG) catalysed by Escherichia coli beta-Galactosidase in the presence of 0-35% w/v 6kD polyethyleneglycol (PEG(6000)). The Eadie-Hofstee data analysis exhibited single straight lines for PNPG at all [PEG(6000)] as well as for ONPG in the absence of PEG(6000) so a Michaelian model was applied to calculate the kinetic parameters K-M and k(cat) (catalytic rate constant) values. However, for ONPG hydrolysis in the presence of PEG(6000), 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 kat 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 PEG(6000). With PNPG, K-M as well as k(cat) varied with [PEG(6000)] like a parabola opening upward with a minimum at 15 %w/v [pEG(6000)]. In the case of ONPG, one of the components became constant while the other component exhibited a slight increasing tendency in k(cat) plus high and [PEG(6000)]-dependent increasing K-M values. Sedimentation velocity analysis demonstrated that PEG(6000) impaired the diffusion of beta-Gal but not that of substrates. In conjunction, kinetic data reflected complex combinations of PEG(6000)-induced effects on enzyme structure, water structure, thermodynamic activities of all the chemical species participating in the reaction and protein diffusion. (C) 2019 Published by Elsevier Inc.