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

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 muM) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37 degreesC was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 muM), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 muM) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: K-m = 12.0 +/- 0.8 muM and k(cat) = 48.4 /1 1.0 min(-1). The data indicate that the inhibition of hK1 by 4-aminobenz amidine and benzamidine is linear competitive, while the inhibition by 4-ni troaniline and aniline is linear mixed, with the inhibitor being able to bi nd 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 benzamidin e, 4-nitroaniline and aniline were 146 +/- 10, 1,098 +/- 91, 38.6 +/- 5.2 a nd 37,340 +/- 5,400 muM, respectively. The calculated K-i' values for 4-nit roaniline and aniline were 289.3 +/- 92.8 and 310,500 +/- 38,600 muM, respe ctively. The fact that K-i'>K-i indicates that 4-nitroaniline and aniline b ind to a second binding site in the enzyme with lower affinity than they bi nd to the active site. The data about the inhibition of hK1 by 4-aminobenza midine and benzamidine help to explain previous observations that esters, a nilides or chloromethyl ketone derivatives of N alpha -substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds.