Low Operational Stability of Enzymes in Dry Organic Solvents: Changes in the Active Site Might Affect Catalysis

Molecules

Vibha Bansal¹  Yamixa Delgado¹  Marc D. Legault¹ 

[1] ¹Department of Chemistry, University of Puerto Rico at Cayey, Cayey, 00736, Puerto Rico ²Department of Chemistry, University of Puerto Rico at Humacao, Humacao, 00791, Puerto Rico ³Department of Physics, University of Puerto Rico at Bayamón, Bayamón, 00959, Puerto Rico

关键词: subtilisin Carlsberg;    organic solvents;    EPR spectroscopy;    enzyme kinetics in organic solvents;   

DOI  :  10.3390/molecules17021870

来源: mdpi

【 摘 要 】

The potential of enzyme catalysis in organic solvents for synthetic applications has been overshadowed by the fact that their catalytic properties are affected by organic solvents. In addition, it has recently been shown that an enzyme’s initial activity diminishes considerably after prolonged exposure to organic media. Studies geared towards understanding this last drawback have yielded unclear results. In the present work we decided to use electron paramagnetic resonance spectroscopy (EPR) to study the motion of an active site spin label (a nitroxide free radical) during 96 h of exposure of the serine protease subtilisin Carlsberg to four different organic solvents. Our EPR data shows a typical two component spectra that was quantified by the ratio of the anisotropic and isotropic signals. The isotropic component, associated with a mobile nitroxide free radical, increases during prolonged exposure to all solvents used in the study. The maximum increase (of 43%) was observed in 1,4-dioxane. Based on these and previous studies we suggest that prolonged exposure of the enzyme to these solvents provokes a cascade of events that could induce substrates to adopt different binding conformations. This is the first EPR study of the motion of an active-site spin label during prolonged exposure of an enzyme to organic solvents ever reported.

【 授权许可】

CC BY   
This is an open access article distributed under the Creative Commons Attribution License (CC BY) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.