Kinetic characterization of rat tissue kallikrein using N alpha-substituted arginine 4-nitroanilides and N alpha-benzoyl-L-arginine ethyl ester as substrates

Abstract

Hydrolysis of seven Nor-substituted L-arginine 4-nitroanilides: benzoyl-arginine p-nitroanilide (Bz-Arg-Nan), tosyl-arginine p-nitroanilide (Tos-Arg-Nan), acetyl-leucyl-arginine p-nitroanilide (Ac-Leu-Arg-Nan), acetyl-phenylalanyl-arginine p-nitroanilide (Ac-Phe-Arg-Nan), benzoyl-phenylalanyl-arginine p-nitroanilide (Bz-Phe-Arg-Nan), tosyl-phenylalanyl-arginine p-nitroanilide (Tos-Phe-Arg-Nan), and D-valyl-leucyl-aginine p-nitroanilide (D-Val-Leu-Arg-Nan), and the N alpha-substituted L-arginine ester: benzoyl-arginine ethyl ester (Bz-Arg-OEt), by rat tissue kallikrein was studied throughout a wide range of substrate concentrations, The enzyme showed a bimodal behavior with all the substrates tested except Tos-Arg-Nan, At low substrate concentrations (10 to 170 mu M for p-nitroanilides and 50 to 190 mu M for Bz-Arg-OEt) the hydrolysis followed Michaelis-Menten kinetics, but at higher substrate concentrations (150 to 700 mu M for p-nitroanilides and 200 to 1800 mu M for Bz-Arg-OEt) a deviation from Michaelis-Menten kinetics was observed with a significant decrease in hydrolysis rates. At high concentrations of the p-nitroanilide substrates, partial enzyme inhibition was observed, whereas complete enzyme inhibition was observed with Bz-Arg-OEt at high concentration, The kinetic parameters reported here were calculated from data for substrate concentrations range where the enzyme followed Michaelis-Menten behavior. D-Val-Leu-Arg-Nan (K-m = 24 +/- 2 mu M; V-max = 10.42 +/- 0.28 mu M/min) was the best substrate tested, followed by Ac-Phe-Arg-Nan (K-m = 13 +/- 2 mu M; V-max = 3.21 +/- 0.11 mu M/min), while Tos-Arg-Nan (K-m = 29 +/- 2 mu M; V-max = 0.10 +/- 0.002 mu M/min) was the worst of the tested substrates for rat tissue kallikrein, For the hydrolysis of Bz-Arg-OEt (K-m = 125 +/- 15 mu M; V-max = 121.3 +/- 7.6 mu M/min), the kinetic parameters using a substrate inhibition model can reasonably account for the observed enzyme behavior, with a K-si value about 13.6 times larger than the estimated K-m value.