When proteins are engineered with a polymer surfactant coating on their surface, they can form a liquid phase by themselves, without the need for a solvent, such as, say, water. However, such solvent-free protein liquids (SFPL), despite their capability to function at temperatures above those in aqueous solutions, exhibit much reduced catalytic rates. A comprehensive understanding of the nature of substrates in such liquids is crucial to reason out the reduced catalytic activity of enzymes as SFPL media, and thus identify the means to improve the same. Employing atomistic molecular dynamics simulations of lipase A from Bacillus subtilis in its SFPL form, we demonstrate that at low concentrations, the substrate molecules are located mostly in the hydrophilic layer of the surfactant shell that ensheaths the enzyme; substrates in this SFPL are present in various conformations with similar propensities as in the aqueous solution. Slower translational diffusion and reorientational dynamics, as well as the reduced tendency of a substrate molecule to closely interact with the enzymes in the SFPL medium have been identified herein as the contributing factors for the reduced activity of enzymes in this hybrid liquid. At high concentrations of substrates corresponding to those used in in vitro experiments, the formation of an enzyme-substrate complex is observed. Microscopic insights reported here can aid in the choice of surfactants to improve the catalytic rate of enzymes in SFPL.
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