The Effect of Thionine on the Kinetics of the β-Trypsin-catalyzed Hydrolysis of Tosyl-l-arginine Methyl Ester

By means of steady state and pre-steady state kinetic experiments in the presence of the acridine dye, thionine, the rate constants associated with formation and disappearance of the presumed acyl enzyme intermediate were determined for the reaction of β-trypsin with tosyl-l-arginine methyl ester. At pH 4.9, 25°, 0.5 m sodium chloride, the kinetic constants are: k23 (acylation rate constant), 200 sec-1; k34 (deacylation rate constant), 0.25 sec-1; K's (enzyme-substrate dissociation constant), 1.25 mm. Evidence is presented that thionine is not bound at the active site of trypsin. A ternary enzyme-substrate-thionine complex exists with the same dissociation constant as the enzyme-substrate complex. An acyl enzyme-thionine complex exists with a dissociation constant higher than the enzyme-thionine complex. The presence of bound thionine slows down the acylation but speeds up the deacylation of trypsin by tosyl-l-arginine methyl ester.