Kinetic Studies at High pH of the Trypsin-catalyzed Hydrolysis of Nα-Benzoyl Derivatives of l-Arginamide, l-Lysinamide, and S-2-Aminoethyl-l-cysteinamide and Related Compounds

A steady state kinetic analysis has been made of the trypsin-catalyzed hydrolysis of Nα-benzyol-l-arginamide, Nα-benzoyl-l-lysinamide, and Nα-benzoyl-S-2-aminoethyl-l-cysteinamide at pH values from 8 to 10.7 at 30°. The Km (app) of the arginine derivative changes from 2.5 to 4.5 x 10-3 m while that of the lysine derivative increases from 4.6 to 17.2 x 10-3 m in going from pH 8 to 10.7. Over the same range the kcat for the arginine derivative decreases from 2.8 to 1.5 sec-1 while the value for the lysine derivative decreases from 1.9 to 1.3 sec-1. At pH 8.0, the Km (app) for the aminoethylcysteine derivative (4.3 x 10-3 m) was about the same as that for lysine derivative whereas the value of kcat of 0.33 sec-1 was considerably lower. At pH 9, 30° and under, the conditions in which the reaction was first order with respect to substrate concentration, the relative rate of cleavage of the arginyl, lysyl, and aminoethylcysteinyl bonds was 100:40:7. From the effect of pH on Km (app) and kcat of the arginine derivative, it was deduced that the enzyme possessed an ionizable group with a pKa of 10.4 which must be protonated in order to have a fully active enzyme. After correcting for the ionization of the enzyme, the effect of pH on the kinetic parameters of lysine and aminoethylcysteine derivatives could be correlated with the dissociation of their distal amino groups. The ionization constants of these groups as determined by the kinetic method agreed within 0.3 pH unit of their values as determined by titration in distilled water. More importantly, it was found by titration that the pKa of the α-amino group of the aminoethylcysteine derivative was 9.4 while the pKa for the e-amino group of the lysine derivative was 10.3. The same difference in the ionization constant was determined by the kinetic method. As a result of the difference in pK values between the basic groups of arginyl, lysyl, and aminoethylcysteinyl residues, it should be possible at high pH (10.7) to catalyze the hydrolysis of arginyl bonds in an aminoethylated protein with no cleavage of aminoethylcysteinyl bonds and with only slight cleavage of lysyl bonds. At pH 7.9, there was no indication of substrate activation by the above mentioned amides or by Nα-tosyl-l-arginamide or Nα-tosyl-l-lysinamide over the substrate range of 5 x 10-5 to 5 x 10-2 m. Under the same conditions the phenomenon attributed to substrate activation was observed for Nα-tosyl-l-arginine methyl ester and Nα-tosyl-l-lysine methyl ester. These studies were performed with trypsin that had been treated with the chymotrypsin inhibitor, l-(1-tosylamido-2-phenyl)ethyl chloromethyl ketone, and subjected to chromatography on carboxymethyl cellulose. The chromatography revealed two active components which were apparently interconvertible on rechromatography.