The inhibition of chymotrypsin by diethyl p-nitrophenyl phosphate

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Abstract
The proteolytic activity of chymotrypsin was inhibited by diethyl p-nitrophenyl phosphate (E600). 50% inhibition was produced by 3 x 10-4 M E600 after 423 min. incubation at pH 7.6 at 25[degree]. The inhibition was irreversible. p-Nitrophenyl ethyl carbonate and p-nitrophenyl acetate were substrates for chymotrypsin. Values of Km and turnover number were obtained. p-Nitrophenol was liberated during inbibition of chymotrypsin by E600. The molar concn. of nitrophenol liberated after complete inhibition was equivalent to the molar concn. of the enzyme. The rates of inhibition by E600 of the proteolytic, amidase and amino acid esterase were equal and corresponded exactly to the rate of liberation of nitrophenol during the inhibition. The kinetics of liberation of nitrophenol were explained on the basis of a bimolecular reaction between enzyme and inhibitor. The velocity constant obtained satisfied the kinetics of inhibition of proteolytic activity. The results supported the hypothesis of a single active center in chymotrypsin.