Soft interaction and excluded volume effect compete as polyethylene glycols modulate enzyme activity

Area of Science:

• Biochemistry
• Physical Chemistry
• Enzyme Kinetics

Background:

• Polyethylene glycols (PEGs) exhibit concentration- and chain-length-dependent interactions with biomolecules, influencing their properties through preferential binding or excluded volume effects.
• Understanding these interactions is crucial for protein stabilization, drug delivery, and biocatalysis applications.
• Hen-egg-white lysozyme (HEWL) is a model enzyme widely used to study enzyme activity and stability under various conditions.

Purpose of the Study:

• To investigate the impact of ethylene glycol (EG) and polyethylene glycols (PEGs) of varying chain lengths (400 and 4000 Da) on the enzymatic activity of HEWL.
• To elucidate the underlying mechanisms, including solvent effects, excluded volume, and molecular interactions, governing HEWL's catalytic efficiency.
• To explore the correlation between HEWL's thermal stability and its activity in the presence of PEGs.

Main Methods:

• Enzyme activity assays were performed using Micrococcus lysodeikticus (M. Lys.) as the substrate to determine the turnover number of HEWL.
• Circular dichroism (CD) and fluorescence spectroscopy were employed to assess changes in protein structure and conformation.
• Thermal unfolding experiments were conducted to determine the melting temperature (T_m) of HEWL in the presence of cosolutes.

Main Results:

• HEWL activity exhibited a bell-shaped profile with increasing PEG concentration, with optimal efficiency observed at low PEG concentrations (e.g., 2% PEG-400).
• Activity decreased at higher PEG concentrations, suggesting a balance between stabilizing and destabilizing effects.
• CD and fluorescence data indicated that solvent polarity, excluded volume, nonspecific interactions, and structural flexibility collectively influence enzyme activity. Thermal unfolding temperature (T_m) also showed a similar bell-shaped trend with PEG concentration.

Conclusions:

• The observed enzyme efficiency profile is a result of competing factors, including excluded volume effects and soft interactions between HEWL and PEG molecules.
• A minimum in the activation energy barrier for catalysis was observed at low osmolyte concentrations.
• The study highlights the complex interplay of solution properties and protein structure in modulating enzyme performance, with potential implications for protein engineering and formulation.