Serine proteinase inhibition by the active site titrant N-alpha-(N,N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester - A comparative study

Kinetics for the hydrolysis of the chromogenic active-site titrant N-alpha- (N,N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester (Dmc-azaOrn- ONp) catalysed by bovine beta-trypsin, bovine alpha-thrombin, bovine Factor Xa, human alpha-thrombin, human Factor Xa, human Lys77-plasmin, human urin ary kallikrein, M-r 33 000 and M-r 54 000 species of human urokinase, porci ne pancreatic beta-kallikrein-A and -B and Ancrod (the coagulating serine p roteinase from the Malayan pit viper Agkistrodon rhodostoma venom) have bee n obtained between pH 6.0 and 8.0, at 21.0 degrees C, and analysed in paral lel with those for the enzymatic cleavage of N-alpha-(N, N-dimethylcarbamoy l)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). The enzyme kinetic s are consistent with the minimum three-step catalytic mechanism of serine proteinases, the rate-limiting step being represented by the deacylation pr ocess. Bovine beta-trypsin kinetics an modulated by the acid-base equilibri um of the His57 catalytic residue (pK(a) approximate to 6.9). Dmc-azaOm-ONp and Dmc-azaLys-ONp bind stoichiometrically to the serine proteinase active site, and allow the reliable determination of the active enzyme concentrat ion between 1.0 x 10(-6) M and 3.0 x 10(-4) M. The affinity and the reactiv ity for Dmc-azaOrn-ONp (expressed by K-s and k(+2)/K-s, respectively) of th e serine proteinases considered are much lower than those for Dmc-azaLys-ON p. The very different affinity and reactivity properties for Dmc-azaOm-ONp and Dmc-azaLys-ONp have been related to the different size of the ornithine /lysine side chains, and to the ensuing different positioning of the active -site titrants upon binding to the enzyme catalytic centre (i.e. to P-1-S-1 recognition). These data represent the first detailed comparative investig ation on the catalytic properties of serine proteinases towards an ornithin e derivative (i.e. Dmc-azaOm-ONp).