title: Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline.
abstract: Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 microM) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37 degrees C was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 microM), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 microM) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 +/- 0.8 microM and k cat = 48.4 +/- 1.0 min(-1). The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 +/- 10, 1,098 +/- 91, 38.6 +/- 5.2 and 37,340 +/- 5,400 microM, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 +/- 92.8 and 310,500 +/- 38,600 microM, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds.
authors.affiliation: Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.
authors.forename: M O
authors.initials: MO
authors.lastname: Sousa
authors.forename: T L
authors.initials: TL
authors.lastname: Miranda
authors.forename: E B
authors.initials: EB
authors.lastname: Costa
authors.forename: E R
authors.initials: ER
authors.lastname: Bittar
authors.forename: M M
authors.initials: MM
authors.lastname: Santoro
authors.forename: A F
authors.initials: AF
authors.lastname: Figueiredo
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry; D000814:Aniline Compounds / Q000494:pharmacology*; D001550:Benzamidines / Q000494:pharmacology*; D001665:Binding Sites; D002863:Chromogenic Compounds / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D016014:Linear Models; D009842:Oligopeptides / Q000378:metabolism*; D010940:Plant Proteins / Q000494:pharmacology; D020840:Tissue Kallikreins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D014361:Trypsin Inhibitors; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
chemical_list: D000814:Aniline Compounds; D001550:Benzamidines; D002863:Chromogenic Compounds; D009842:Oligopeptides; D010940:Plant Proteins; D014361:Trypsin Inhibitors; C019498:4-nitroaniline; C006500:4-aminobenzamidine; C031006:Val-Leu-Arg-p-nitroanilide; D000516:alpha-Amylases; D020840:Tissue Kallikreins; D000581:Amidohydrolases