Enzymatic catalysis in nonaqueous solvents.

TLDR

Subtilisin and alpha‑chymotrypsin act as powerful catalysts in dry organic solvents, where transesterification follows Michaelis–Menten kinetics that correlate with solvent hydrophobicity, and their activity depends on minimal water, solvent‑induced water stripping, and protein rigidity that prevents unfolding. In octane, chymotrypsin and subtilisin achieve ~100 billion‑fold rate enhancements, retain activity unless covalently modified at the active site, reverse specificity toward competitive inhibitors, and exhibit markedly improved thermal and storage stability compared to water.

Abstract

Subtilisin and alpha-chymotrypsin vigorously act as catalysts in a variety of dry organic solvents. Enzymatic transesterifications in organic solvents follow Michaelis-Menten kinetics, and the values of V/Km roughly correlate with solvent's hydrophobicity. The amount of water required by chymotrypsin and subtilisin for catalysis in organic solvents is much less than needed to form a monolayer on its surface. The vastly different catalytic activities of chymotrypsin in various organic solvents are partly due to stripping of the essential water from the enzyme by more hydrophilic solvents and partly due to the solvent directly affecting the enzymatic process. The rate enhancements afforded by chymotrypsin and subtilisin in the transesterification reaction in octane are of the order of 100 billion-fold; covalent modification of the active center of the enzymes by a site-specific reagent renders them catalytically inactive in organic solvents. Upon replacement of water with octane as the reaction medium, the specificity of chymotrypsin toward competitive inhibitors reverses. Both thermal and storage stabilities of chymotrypsin are greatly enhanced in nonaqueous solvents compared to water. The phenomenon of enzymatic catalysis in organic solvents appears to be due to the structural rigidity of proteins in organic solvents resulting in high kinetic barriers that prevent the native-like conformation from unfolding.