Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline

• Main Authors: M.O. Sousa, T.L.S. Miranda, E.B. Costa, E.R. Bittar, M.M. Santoro, A.F.S. Figueiredo
• Format: Article
• Language: English
• Published: Associação Brasileira de Divulgação Científica 2001-01-01
• Series: Brazilian Journal of Medical and Biological Research
• Subjects: kinetics of human tissue kallikrein inhibition; tissue kallikrein; 4-nitroaniline; aniline; benzamidine; 4-aminobenzamidine; enzyme inhibition
• Online Access: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2001000100004

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 µM) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37ºC was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 µM), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 µM) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 ± 0.8 µM 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 µM, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 ± 92.8 and 310,500 ± 38,600 µM, 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.