Synthesis and hydrolysis by arginyl-hydrolases of p-nitroanilide chromogenic substrates containing polyethylene glycol and D-gluconyl moieties.

D-Gluconic acid and alpha-carboxymethyl-polyethylene-glycol-omega-methyl ether (PEG) (mol wt 550) were covalently bound at N alpha-amino group of H-Phe-Arg-pNa to study the effect on hydrolysis by arginyl-hydrolases of chromogenic substrates containing high hydrophilic and amphiphilic groups. For comparison, epsilon-aminocaproyl-, sarcosyl- and succinyl-Phe-Arg-pNa were also synthesized. The obtained compounds were assayed as substrates for porcine pancreatic kallikrein, horse urinary kallikrein, tonin and beta-trypsin. Both PEG- and gluconyl-Phe-Arg-pNa had kcat values of hydrolysis 2-4 times higher than the N-acetyl derivative for all the studied enzymes. epsilon-NH2caproyl-Phe-Arg-pNa resulted in the best chromogenic substrate described for the two tissue kallikreins. The PEG-derivative and D-gluconyl groups were also introduced in the N alpha-amino group of H-Arg-pNa and assayed as beta-trypsin substrates. In comparison with benzoyl-Arg-pNa, the D-gluconyl group had no effect on Km but reduced the kcat value more than 15 times; however, PEG-Arg-pNa was hydrolyzed with similar Km but with kcat 5 times higher. The presence of D-gluconyl and PEG groups in the chromogenic substrate molecules increased their water solubility significantly.

Substrate activation of porcine pancreatic kallikrein by N alpha derivatives of arginine 4-nitroanilides.

Hydrolysis of several N alpha-substituted L-arginine 4-nitroanilides with porcine pancreatic kallikrein was studied under different conditions of pH, temperature, and salt concentration. At high substrate concentrations a deviation from Michaelis-Menten kinetics was observed with a significant increase in the hydrolysis rates of almost all substrates. Kinetic data were analyzed on the assumption that porcine pancreatic kallikrein presents an additional binding site with lower affinity for the substrate. Binding to this auxiliary site gives rise to a modulated enzyme species which can hydrolyze an additional molecule of the substrate through a second catalytic pathway. The values of both Michaelis-Menten and catalytic rate constants were higher for the modulated species than for the free enzyme, suggesting a mechanism of enzyme activation by substrate. Kinetic data indicated similar substrate requirements for binding at the primary and auxiliary sites of the enzyme. Tris(hydroxymethyl)aminomethane hydrochloride and NaCl were shown to alter the kinetic parameters of the hydrolysis of N alpha-acetyl-L-Phe-L-Arg 4-nitroanilide by porcine pancreatic kallikrein but not the enzyme activation pattern (ratio of the catalytic constants for the activated and the free enzyme forms). Similar observations were made when the hydrolysis of D-Val-L-Leu-L-Arg 4-nitroanilide was studied under different pH and temperature conditions.

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.