Presteady State Kinetics of Trypsin-Catalyzed Hydrolyses of Dansyl-Arginine Derivatives

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Abstract
Interactions between trypsin and each of five dansyl-arginine derivatives, dansyl-L-arginine methyl ester (L-DAME), dansyl-D-arginine methyl ester (D-DAME), dansyl-L-arginine amide (L-DAA), dansyl-L-arginine (L-DA), and dansyl-D-arginine (D-DA), are accompanied by a fluorescence intensity change which can be followed by the stopped-flow method. These compounds are substrates or products in trypsin-catalyzed hydrolysis reactions. All of these compounds, except L-DAA, show a considerable fluorescence intensity increase in the reaction with trypsin. The observed rate constant, τ−1obsd, for the initial fluorescence intensity enhancement, in the reaction between trypsin and D-DAME yields a typical hyperbolic curve when the rate is plotted as a function of the ligand concentration. This result is consistent with a two-step mechanism (1) in which a fast bimolecular association process is followed by a slower unimolecular isomerization process. The isomerization process may be considered to be associated with a conformational change of the enzyme molecule, induced by the formation of the enzyme-substrate complex (1). The rate of the isomerization process depends on pH. The rates obtained for L-DAME and D-DAME increase linearly with decrease of the hydrogen ion concentration in the pH range below neutral.

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