CALORIMETRIC EVALUATION OF ENZYME-KINETIC PARAMETERS

The measurement of kinetic parameters (k(cat), K(m), K(i)) for a wide range of proteolytic enzymes is vital to contemporary bioorganic and m edicinal chemistry. Enzyme assays based on changes in optical properti es of the system or changes in concentration of an ion detectable elec trochemically are not viable for many enzyme-catalyzed reactions, incl uding proteases and peptidases. Hydrolysis of an amide bond produces n o change in the optical properties or pH of the reaction solution, and as a result no general direct method for the evaluation of protease k inetics exists using underivatized substrates. We report here a microc alorimetric assay which provides a general and straightforward techniq ue for the measurement of kinetic parameters of hydrolysis of underiva tized peptide substrates by proteases. Using this technique, k(cat) va lues as high as 10(5) s-1 can be easily measured. We demonstrate the u tility of the technique by measuring the kinetics of hydrolysis of sev eral N-acylamino acids by the synthetically useful enzyme hog kidney a cylase and the hydrolysis of tetrapeptide p-nitrophenyl anilides by su btilisin BPN'. Although we have used the technique to monitor amide bo nd hydrolysis, the methodology is applicable to any system with approp riate kinetic and thermodynamic properties.