Kinetic analysis of the mechanism for subtilisin in essentially anhydrous organic solvents |
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Authors: | Sudipta Chatterjee and Alan J Russell |
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Institution: | Center for Biotechnology and Bioengineering and Department of Chemical Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania, USA |
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Abstract: | Steady-state kinetic analysis has been used to confirm the catalytic mechanism of lyophilized subtilisin suspended in a variety of organic solvents. Specifically, this article demonstrates that partial reactions can occur between subtilisin and ester substrates in organic solvents. Partitioning of common intermediates between competing acceptors at a constant ratio of products has also been described. The decomposition of a common intermediate formed from different substrates at the same rate is also further evidence of an acyl-enzyme mechanism for subtilisin suspended in anhydrous solvents. Partitioning of a common intermediate to give two products at a constant total rate, and saturation kinetics at varying substrate concentrations, complete a kinetic investigation of the enzyme mechanism. All the data generated support the formation of a stable acyl enzyme during the transesterification reaction catalzyed by subtilisin in the solvents used. |
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Keywords: | Enzymes organic solvents mechanism subtilisin microscopic rate constants active site titration |
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