Potent inactivator of alpha-chymotrypsin: 2,2-dimethyl-3-(N-4-cyanobenzoyl)amino-5-phenyl pentanoic anhydride.

Abstract We synthesized a novel potent α-chymotrypsin inactivator, 2,2-dimethyl-3-( N -4-cyanobenzoyl) amino-5-phenyl pentanoic anhydride, which fulfilled the criteria of a mechanism-based inactivator: first-order kinetics, irreversibility, saturation kinetics and substrate protection. The inactivation rate constant (k inact ) and the enzyme-inhibitor dissociation constant (K I ) were calculated to be 0.017s −1 and 0.071μM, respectively (k inact /K I = 242000 M −1 s −1 ). These kinetic parameters indicate that this compound is one of the most powerful α-chymotrypsin inactivators ever reported. The average number of α-chymotrypsin turnovers per inactivation (partition ratio) was calculated to be 1, which indicates that it is a stoichiometrically ideal inactivator of α-chymotrypsin. We compared the IC50 values of this compound with those of several chymotrypsin-like serine proteinases (bovine α-chymotrypsin, recombinant human chymase and human neutrophil cathepsin G) and a metallo proteinase, rabbit angiotensin converting enzyme (ACE). Our compound, 2,2-dimethyl-3-( N -4-cyanobenzoyl) amino-5-phenyl pen- tanoic anhydride, inhibited bovine α-chymotrypsin potently (IC50 = 1.0 (± 0.2) × 10 −9 M) as well as other chymotrypsin-like serine proteinase; recombinant human chymase (IC50 = 7.0 (± 1.0) × 10 −8 M) and human neutrophil cathepsin G (IC50 = 1.8 (± 0.2) × 10 −7 M). However, rabbit ACE was not inhibited by this compound (IC50 > 1 × 10 −4 M).