Kinetic characterization of rat tissue kallikrein using N alpha-substituted arginine 4-nitroanilides and N alpha-benzoyl-L-arginine ethyl ester as substrates.
Hydrolysis of seven N alpha-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-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 microM for p-nitroanilides and 50 to 190 microM for Bz-Arg-OEt) the hydrolysis followed Michaelis-Menten kinetics, but at higher substrate concentrations (150 to 700 microM for p-nitroanilides and 200 to 1800 microM 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 +/- 2 microM; Vmax = 10.42 +/- 0.28 microM/min) was the best substrate tested, followed by Ac-Phe-Arg-Nan (Km = 13 +/- 2 microM; Vmax = 3.21 +/- 0.11 microM/min), while Tos-Arg-Nan (Km = 29 +/- 2 microM; Vmax = 0.10 +/- 0.002 microM/min) was the worst of the tested substrates for rat tissue kallikrein. For the hydrolysis of Bz-Arg-OEt (Km = 125 +/- 15 microM; Vmax = 121.3 +/- 7.6 microM/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.
Sousa
M O
MO
Departamento de Análises Clínicas e Toxicológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.
Rodrigues
C V
CV
Pena
H B
HB
Alvarenga
M G
MG
Machado-Coelho
G L
GL
Santoro
M M
MM
Juliano
M A
MA
Juliano
L
L
Figueiredo
A F
AF
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D006868:Hydrolysis; D007610:Kallikreins / Q000302:isolation & purification / Q000493:pharmacokinetics* / Q000652:urine; D051381:Rats; D015219:Substrate Cycling
D001120:Arginine; C012208:benzoyl L-arginine methyl ester; D007610:Kallikreins