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Determination of equilibrium and individual rate constants for subtilisin‐catalyzed transesterification in anhydrous environments
Author(s) -
Chatterjee Sudipta,
Russell Alan J.
Publication year - 1992
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260400910
Subject(s) - anhydrous , subtilisin , nucleophile , chemistry , transesterification , catalysis , solvent , equilibrium constant , substrate (aquarium) , reaction rate constant , organic chemistry , enzyme , kinetics , inorganic chemistry , oceanography , physics , geology , quantum mechanics
We report here the first determinations of individual rate constants and equilibrium constants for enzymatic reactions in essentially anhydrous organic solvents. Using the added nucleophile method we have measured the effect of changing solvent on the binding and catalytic steps for subtilisin‐catalyzed transesterification of N ‐protected amino acid esters. The detailed information generated indicates that once the substrate has bound to the enzyme, the catalytic machinery can work at rates equivalent to those in water. The decreased overall rates for subtilisin suspended in anhydrous solvents are merely the result of extremely high values for K s , in most cases, coupled with low concentrations of nucleophile (∼1.0 M in organic solvents, and 55 M in water). The method described, which is generally applicable, and straightforward experimentally, will, we believe, enable a clearer understanding of how changing solvent can predictably affect the activity and specificity of the enzyme. © 1992 John Wiley & Sons, Inc.