Synthesis and kinetic parameters of hydrolysis by trypsin of some acyl-arginyl-p-nitroanilides and peptides containing arginyl-p-nitroanilide.

Four acyl-arginyl-p-nitroanilides, nine acetyl-(or benzoyl)-aminoacyl-arginyl-p-nitroanilides and twelve acyl-(or free alpha-amino-)dipeptidyl-arginyl-p-nitroanilides were synthesized, and the kinetic parameters for tryptic hydrolysis of these substrates were determined in 100 mM Tris-HCl buffer, pH 8.0, containing 10 mM CaCl2 at 37 degrees C. Among the acyl-arginyl-p-nitroanilides, Octanoyl-Arg-pNA was hydrolyzed four times more rapidly by trypsin than the commonly used substrate Benzoyl-Arg-pNa. The best trypsin substrates contain proline and norleucine at subsite P2, indicating that unbranched aliphatic side chain folded as the beta, gamma and delta methylenes are in proline provides the most favorable conditions for S2P2 interaction. Extending the length of the substrates from di- to tripeptidyl-pNA did not have a large influence on the kinetic parameters. However, Phe at the P3 position had a clear favorable effect, in contrast to Pro, which is unfavorable only when the benzoyl group is present at P4. The series Ac-Phe (or D-Phe)-Gly-Arg-pNA and Phe (or D-Phe)-Gly-Arg-pNA were studied. The benzyl side chain of D-Phe has a more favorable interaction at S3 than Phe. A P4 - CO. . .HN-S4 hydrogen bond is proposed to stabilize P3/S3 interaction when an acetyl group is present on the alpha-amino group of the Phe residue, and the reverse would be expected to occur for the corresponding D-epimer.