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

M.O. Sousa, T.L.S. Miranda, E.B. Costa, E.R. Bittar, M.M. Santoro, A.F.S. Figueiredo
2001 Brazilian Journal of Medical and Biological Research  
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 37 o C 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: K m = 12.0 ± 0.8 µM and k cat = 48.4 ± 1.0 min -1 . The data indicate that the inhibition of hK1 by
more » ... nhibition 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 K i yielding an EI complex, and to the ES complex with a dissociation constant K i , yielding an ESI complex. The calculated K i 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 K i values for 4nitroaniline and aniline were 289.3 ± 92.8 and 310,500 ± 38,600 µM, respectively. The fact that K i >K i 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 Na-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds.
doi:10.1590/s0100-879x2001000100004 pmid:11151026 fatcat:75xwcndo65em3ads3vonibk6na