A new method for the determination of the amidase activity of trypsin: kinetics of the hydrolysis of benzoyl-l-arginineamide

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Abstract
Although formaldehyde alters the chemical structure of trypsin it has no effect on the activity of the enzyme at the pH optimum. A method for following the trypsin-catalyzed hydrolysis of amides based on the above observation is described. The enzyme-substrate dissociation constant for benzoyl-L-arginineamide was determined (Km = 3.1 x 10-3M) as well as the first-order specific rate of decomposition of the enzyme-substrate complex (k3 = 0.043 sec.-1). The pronounced deceleration of trypsin-catalyzed hydrolysis with time was explained in terms of the self-digestion of the enzyme (a process which is inhibited by the substrate).