Mammalian Tissue Trypsin-like Enzymes: Substrate Specificity and Inhibitory Potency of Substituted Isocoumarin Mechanism-Based Inhibitors, Benzamidine Derivatives, and Arginine Fluoroalkyl Ketone Transition-State Inhibitors

Amino acid and peptide thioesters which contained Arg or Lys in the P1 position were tested as substrates for rat skin tryptase, and the kinetic constants kcat/KM for the better substrates such as Z-Aba-Arg-SBzl, and Z-Gly-Arg-SBzl were over 5,000,000 M−1 s−1. The inhibitory potency of arginine fluoroalkyl ketones, benzamidine derivatives, and substituted isocoumarins containing basic functional groups was studied with rat skin tryptase, human lung tryptase, human skin tryptase, and bovine trypsin. 1-Naphthoyl-Arg-CF3 was the best arginine fluoroalkyl ketone reversible inhibitor for rat skin tryptase with a KI of 0.9 μM. 1-(4-Amidinophenyl)-3-(4-phenoxyphenyl) urea showed competitive inhibition against bovine trypsin and rat skin tryptase with KI values of 2 and 4 μM, respectively. Isocoumarin derivatives with isothioureidoalkoxy substituents at the 3 position were potent irreversible inhibitors of these three tryptases with kobs/[I] values of 104-105 M−1 s−1. 4-Chloro-3-(2-isothioureido)ethoxy-7-phenylcarbamoylaminoisocoumarin and 7-benzylcarbamoylamino-4-chloro-3-(3-isothioureido)propoxyisocoumarin inactivated trypsin and formed stable trypsin-inhibitor complexes which regained less than 8% of activity upon standing in the pH 7.5 buffer and regained 30-75% of activity in the presence of 0.3 M NH2OH after 1 day, In contrast, the complexes with rat skin tryptase regained activity rapidly, indicating differences in the inhibition mechanism and active site structures of these related enzymes.