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

https://doi.org/10.1016/j.bbrc.2019.05.081Get rights and content

Highlights

  • PEG induced a biphasic kinetics in the β-Gal catalysed hydrolysis of ONPG.

  • ONPG hydrolysis detects energies or availabilities of H2O populations as reactants.

  • The rate limiting step of PNPG hydrolysis is insensitive to H2O populations diversity.

  • PEG impaired the diffusion β-Galactosidase but not of the substrates.

  • 5β-Gal catalytic activity exhibits PEG-dependent mixed diffusion-reaction control.

Abstract

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.

Introduction

Enzymatic activities were historically assayed in dilute solutions where macromolecular crowding (MC), molecular confinement and their consequences were not taken into account.

Environments with MC are characterized by large surface areas in contact with the solvent. This produces a solvent structure perturbation that can affect protein conformation with the consequent effects on the catalytic activity [1]. In the case of β-Gal, steady state fluorescence and Fourier transformed infrared spectroscopic observations were compatible with MC-induced disordering and restricted internal dynamics as a result of excluded volume and solvent structuring effects. This leaded to a non-optimal substrate-binding site and a less conformationally strained protein [2].

Water activity (aw) and solvent ordering can also affect the enzyme kinetics through its effect on the hydrophobic interaction between the enzyme and the substrate [3,4] as well as modifying the thermodynamic activities of substrates, enzyme and transition state intermediaries (ai) [5]. Importantly for hydrolytic reactions, water structuring may affect its availability as reagent. The latter may be mainly expected when water takes part of the rate limiting step of a reaction. This is the case of the enzymatic hydrolysis mechanism of ortho-nitrophenyl-β-D-galactopyranoside (ONPG), but not of its para- isomer (PNPG) [6]. It is worth to note that differential kinetic mechanisms for both substrates when the enzyme was confined in a silicate matrix where several water populations characterized by their molecular mobility could be identified [4].

Polyethyleneglycol (PEG) is often used to simulate MC. In a previous work from our group, spin-lattice relaxation time (T1) obtained from 1H NMR experiments in PEG-water systems let us find a close relation between the dynamics of PEG and water [7]. Thus, we identified two contributing components in each proton system, PEG and water, presenting values of T1 with very different orders of magnitude. The shorter 1H-T1 values associated to water and PEG exhibited an approximate matching. Based on this information we concluded that there exists a network of interactions (hydrogen bonds) between the solute and the solvent. This resulted in the presence of an ordered and dehydrated structure of PEG folded and/or self-assembled in equilibrium with a more flexible monomer structure with the ability to affect the global hydrogen bond network of the entire aqueous solution.

Here we got deeply in the question of how PEG-induced MC tunes catalytic parameters for the tetrameric β-Galactosidase (β-Gal) from Escherichia coli and affects the reaction mechanism. We focused in water as a reagent and PEG as not only a water structuring agent but also volume excluding macromolecule which could affect the diffusion of the chemical species taking place in the reaction (enzyme, substrates and products). Hence we analyzed the hydrolysis not only of ONPG as we did before, but also of PNPG, which reaction mechanism does not involve water in the rate limiting step, in the presence of PEG6000 as MC agent. The kinetic model that best fitted the experimental results in each case was applied. The effect of MC on β-Gal hydrodynamic behavior was analyzed using analytical ultracentrifugation.

Taken together present and previous results let us conclude that MC modulates kinetic data in a way that accompanies the complex combinations between PEG6000-induced effects on protein diffusion, enzyme structure, water structure and thermodynamic activities of all the chemical species participating in the reaction.

Section snippets

Materials

The enzyme β-Gal from E. coli [EC3.2.1.23] Grade VII (specific activity 600-1200 UI/mg protein), 2-nitrophenol (ONP) and the substrates 2-nitrophenyl-β-D-galactopyranoside (ONPG) and 4-nitrophenyl-β-D-galactopyranoside (PNPG) were obtained from Sigma Chem. Co. (St. Louis, MO). PEG6000 was from Anedra. Other reagents and solvents were of analytical grade.

Enzymatic activity determination

The hydrolysis reaction catalyzed by β-Gal was studied with each of two substrates, ONPG and PNPG within concentration ranges 0.05×10−3 - 2.0×10

Effect of the molecular crowding on the β-Gal catalysed PNPG and ONPG hydrolysis

β-Gal enzymatic activity was assayed in the absence and in the presence of increasing concentrations of the crowding agent (CA) PEG6000, with ONPG and PNPG as substrates.

The Eadie-Hofstee data analysis of PNPG hydrolysis (Fig. 1a) showed single straight lines. This supported a Michaelian behavior for the hydrolysis of this substrate within the whole concentration range and all the experimental conditions studied as demonstrated by the initial reaction rate (V0) vs. substrate concentration

Acknowledgements

This work was partially financed by Foncyt, Mincyt-Córdoba, SeCyT-Universidad Nacional de Córdoba and CONICET from Argentina. All authors are members of the later institution. MVN, MIB and MAP are professors of Universidad Nacional de Córdoba. Sedimentation velocity analysis were performed in the Analytical Ultracentrifugation Laboratory form IIBYT(CONICET-UNC).

References (20)

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