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

ABSTRACT

Hydrolysis of seven N(alpha-substituted L-arginine 4-nitroanilides henzoyl-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-arginine 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 muM for p-nitroanilides and 50 to 190 muM for Bz-Arg-OEt) the hydrolysis followed Michaelis-Menten kinetics, but at higher substrate concentrations (150 to 700 muM for p-nitroanilides and 200 to 1800 muM 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 (Km = 24 ñ 2 muM; Vmax 10.42 ñ 0.28 muM/min) was the best substrate tested, followed by Ac-Phe-Arg-Nan (Km = 13 ñ 2 muM; Vmax = 3.21 ñ 0.11 muM/min), while Tos-Arg-Nan (Km = 29 ñ 2 muM; Vmax, = 0. 10 ñ 0.002 muM/min) was the worst of the tested substrates for rat tissue kallikrein. For the hydrolysis of Bz-Arg-OEt (Km = 125 ñ 15 muM; Vmax = 121.3 ñ 7.6 muM/min), the kinetic parameters using a substrate inhibition model can reasonably account for the observed enzyme behavior, with a Ksi value about 13.6 times larger than the estimated Km value.