Journal of Organic Chemistry p. 3507 - 3510 (1993)
Update date:2022-08-16
Topics:
Williams, Brent A.
Toone, Eric J.
The measurement of kinetic parameters (kcat, Km, Ki) for a wide range of proteolytic enzymes is vital to contemporary bioorganic and medicinal chemistry.Enzyme assays based on changes in optical properties of the system or changes in concentration of an ion detectable electrochemically are not viable for many enzyme-catalyzed reactions, including proteases and peptidases.Hydrolysis of an amide bond produces no change in the optical properties or pH of the reaction solution, and as a result no general direct method for the evaluation of protease kinetics exists using underivatized substrates.We report here a microcalorimetric assay which provides a general and straightforward technique for the measurement of kinetic parameters of hydrolysis of underivatized peptide substrates by proteases.Using this technique, kcat values as high as 105 s-1 can be easily measured.We demonstrate the utility of the technique by measuring the kinetics of hydrolysis of several N-acylamino acids by the synthetically useful enzyme hog kidney acylase and the hydrolysis of tetrapeptide p-nitrophenyl anilides by subtilisin BPN'.Although we have used the technique to monitor amide bond hydrolysis, the methodology is applicable to any system with appropriate kinetic and thermodynamic properties.
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