title: Substrate activation of porcine pancreatic kallikrein by N alpha derivatives of arginine 4-nitroanilides.
abstract: Hydrolysis of several N alpha-substituted L-arginine 4-nitroanilides with porcine pancreatic kallikrein was studied under different conditions of pH, temperature, and salt concentration. At high substrate concentrations a deviation from Michaelis-Menten kinetics was observed with a significant increase in the hydrolysis rates of almost all substrates. Kinetic data were analyzed on the assumption that porcine pancreatic kallikrein presents an additional binding site with lower affinity for the substrate. Binding to this auxiliary site gives rise to a modulated enzyme species which can hydrolyze an additional molecule of the substrate through a second catalytic pathway. The values of both Michaelis-Menten and catalytic rate constants were higher for the modulated species than for the free enzyme, suggesting a mechanism of enzyme activation by substrate. Kinetic data indicated similar substrate requirements for binding at the primary and auxiliary sites of the enzyme. Tris(hydroxymethyl)aminomethane hydrochloride and NaCl were shown to alter the kinetic parameters of the hydrolysis of N alpha-acetyl-L-Phe-L-Arg 4-nitroanilide by porcine pancreatic kallikrein but not the enzyme activation pattern (ratio of the catalytic constants for the activated and the free enzyme forms). Similar observations were made when the hydrolysis of D-Val-L-Leu-L-Arg 4-nitroanilide was studied under different pH and temperature conditions.
authors: Department of Biochemistry, Escola Paulista de Medicina, São Paulo, Brazil.
authors: L
authors: L
authors: Oliveira
authors: M S
authors: MS
authors: Araujo-Viel
authors: L
authors: L
authors: Juliano
authors: E S
authors: ES
authors: Prado
mesh_terms: D000813:Anilides / Q000378:metabolism*; D000818:Animals; D004789:Enzyme Activation; D007610:Kallikreins / Q000378:metabolism*; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology*; D013379:Substrate Specificity; D013552:Swine; D013816:Thermodynamics
chemical_list: D000813:Anilides; D009842:Oligopeptides; D007610:Kallikreins