Kinetics for the hydrolysis of the chromogenic active-site titrant N(α)-(N,N-dimethylcarbamoyl)-α-azaornithine p-nitrophenyl ester (Dmc- azaOrn-ONp) catalysed by bovine β-trypsin, bovine α-thrombin, bovine Factor Xa, human α-thrombin, human Factor Xa, human Lys77-plasmin, human urinary kallikrein, M(r) 33 000 and M(r) 54 000 species of human urokinase, porcine pancreatic β-kallikrein-A and -B and Ancrod (the coagulating serine proteinase from the Malayan pit viper Agkistrodon rhodostoma venom) have been obtained between pH 6.0 and 8.0, at 21.0 °C, and analysed in parallel with those for the enzymatic cleavage of N(α)-(N,N-dimethylcarbamoyl)α-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). The enzyme kinetics are consistent with the minimum three-step catalytic mechanism of serine proteinases, the rate- limiting step being represented by the deacylation process. Bovine β-trypsin kinetics are modulated by the acid-base equilibrium of the His57 catalytic residue (pK(a) ≃ 6.9). Dmc-azaOrn-ONp and Dmc-azaLys-ONp bind stoichiometrically to the serine proteinase active site, and allow the reliable determination of the active enzyme concentration between 1.0 x 10- 6 M and 3.0 x 10-4 M. The affinity and the reactivity for Dmc-azaOrn-ONp (expressed by K(s) and k+2/K(s), respectively) of the serine proteinases considered are much lower than those for Dmc-azaLys-ONp. The very different affinity and reactivity properties for Dmc-azaOrn-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 P1-S1 recognition). These data represent the first detailed comparative investigation on the catalytic properties of serine proteinases towards an ornithine derivative (i.e. Dmc- azaOrn-ONp).
- Enzyme inhibition
- Enzyme-substrate/inhibitor recognition
- Trypsin-like serine proteinase
ASJC Scopus subject areas