Abstract
The trypsin-catalyzed hydrolysis of a synthetic ester substrate, dansyl-n-arginine methyl ester (D-DAME), was followed by the entire progression curve method. This ester substrate, which is a D-enantiomer of arginine, is hydrolyzed to the extent of nearly 100 % without causing substrate activation. The experiments were performed by changing either the pH or the initial substrate concentration. The double-reciprocal plot of the velocity of hydrolysis against the residual substrate concentration showed good linearity. As regards the dependency of this straight line on pH at a constant initial substrate concentration, both the slope and the intercept on the 1/v axis decreased with decrease of hydrogen ion concentration in the pH range below 8 and the lines intersected at a point in the second quadrant. At constant pH the slope of the double-reciprocal plot between the velocity of hydrolysis and the residual substrate concentration increased linearly with increase of the initial substrate concentration. The results obtained cannot be interpreted simply in terms of the three-step mechanism for the trypsinand chymotrypsin-catalyzed hydrolyses of ester substrates. In particular, it is significant that the catalytic rate constant obtained for the overall enzymatic reaction is very similar to the rate constant of conformational change of the enzyme molecule due to the formation of enzyme-substrate complex in the trypsin-catalyzed hydrolyses of L-and D-DAME.
