Presteady state kinetics of trypsin-catalyzed hydrolyses of dansyl-arginine derivatives.
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, tau obsd -1, 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.
D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D003619:Dansyl Compounds / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism*
D003619:Dansyl Compounds; D001120:Arginine; D014357:Trypsin