Catalysis by chymotrypsinogen. Demonstration of an acyl-zymogen intermediate

Bovine chymotrypsinogen reacts stoichiometrically with diisopropyl phosphorofluoridate (DFP) and catalyzes the hydrolysis of the ester p-nitrophenyl p-guanidinobenzoate (NPGB). These reactions are mutually exclusive, indicating that the same residue on the active site (Ser-195) is involved in both cases. Although the second-order rate of ester hydrolysis by chymotrypsinogen is 10⁶-10⁷ times slower than by chymotrypsin, the reaction proceeds in both cases via the formation of acyl intermediates. The deacylation rate of the isolated acyl-enzyme is only 70 times slower than that of acylenzyme and is dependent upon the ionization of a single group, presumably His 57. The hydrolysis of NPGB by the homologous zymogen-enzyme pair, trypsinogen-trypsin, is competitively inhibited by p-aminobenzamidine. The apparent inhibition constant K_i is almost four orders of magnitude higher for the zymogen than for the enzyme. These data suggest that the inferior catalytic properties of the zymogens of the pancreatic serine proteases are primarily due to an undeveloped binding site and only secondarily to a less efficient catalytic apparatus. Circular dichroic spectra of acyl-chymotrypsinogen at pH 4.0 indicate that the changes in ellipticity in the 220-250-nm range induced by acylation resemble those induced in the enzyme. In the range of 260-290 nm, however, the spectrum of the acyl-zymogen is significantly different, indicating a perturbed environment of the pguanidinobenzoyl group and suggesting a different and possibly less efficient mode of binding. These spectral changes are completely reversible upon deacylation.