Journal
BIOTECHNOLOGY AND BIOENGINEERING
Volume 99, Issue 1, Pages 9-17Publisher
WILEY-BLACKWELL
DOI: 10.1002/bit.21510
Keywords
bioconjugates; covalent modification; enzyme activity; FTIR; H/D exchange; non-aqueous enzymology; polyethylene glycol; protein dynamics; subtilisin Carlsberg
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Funding
- NATIONAL CENTER FOR RESEARCH RESOURCES [P20RR016470] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [S06GM008216, S06GM008102] Funding Source: NIH RePORTER
- NCRR NIH HHS [P20 RR-016470] Funding Source: Medline
- NIGMS NIH HHS [S06 GM-08102, S06 GM-08216] Funding Source: Medline
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The employment of enzymes as catalysts within organic media has traditionally been hampered by the reduced enzymatic activities when compared to catalysis in aqueous solution. Although several complementary hypotheses have provided mechanistic insights into the causes of diminished activity, further development of biocatalysts would greatly benefit from effective chemical strategies (e.g., PEGylation) to ameliorate this event. Herein we explore the effects of altering the solvent composition from aqueous buffer to 1,4-dioxane on structural, dynamical, and catalytic properties of the model enzyme subtilisin Carlsberg (SBc). Furthermore, we also investigate the effects of dissolving the enzyme in 1,4-dioxane through chemical modification with poly(ethylene)-glycol (PEG, M-W = 20 kDa) on these enzyme properties. In 1,4-dioxane a 10(4)-fold decrease in the enzyme's catalytic activity was observed for the hydrolysis reaction of vinyl butyrate with D2O and a 50% decrease in enzyme structural dynamics as evidenced by reduced amide H/D exchange kinetics occurred. Attaching increasing amounts of PEG to the enzyme reversed some of the activity loss. Evaluation of the structural dynamic behavior of the PEGylated enzyme within the organic solvent revealed an increase in structural dynamics at increased PEGylation. Correlation analysis between the catalytic and structural dynamic parameters revealed that the enzyme's catalytic activity and enantios-elcctivity depended on the changes in protein structural dynamics within 1,4-dioxane. These results demonstrate the importance of protein structural dynamics towards regulating the catalytic behavior of enzymes within organic media.
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