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 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 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 4-nitroaniline 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.

Are improper kinetic models hampering drug development?

“…So competitive inhibition that is believed to be the predominant way in which inhibitors affect substrate affinity is not able to account for inhibitor interactions that alter the spatial conformation of the active site. An example of this false competitive inhibition is the inhibition of the peptidase kallikrein by benzamidine (Sousa et al, 2001). Inhibition of kallikrein by benzamidine is known to occur when benzamidine blocks the binding of peptide side chains but not the active site (Bernett et al, 2002).…”

Peer Review #2 of "Comparing enzyme activity modifier equations through the development of global data fitting templates in Excel (v0.2)"

“…This could occur through alosteric interactions or even through partial blockade of the active site when the enzyme is associated with the inhibitor. For example, the peptidase kallikrein was believed to be competitively inhibited by benzamidine (Sousa et al, 2001). However, the crystal structure of benzamidine binding to kallikrein demonstrated that it does not block the catalytic site of the enzyme 12PeerJ reviewing PDF | (2017:07:19256:3:0:NEW 2 Nov 2018)…”

Untitled

“…So competitive inhibition which is viewed as the predominant way in which inhibitors affect substrate affinity is not able to account for inhibitor interactions that alter the spatial conformation of the active site. An example of this false competitive inhibition is the inhibition of Kallikrein by benzamidine (Sousa et al, 2001). Inhibition of Kallikrein by benzamidine is known to result from benzamidine blocking the binding of peptide side chains but not the active site (Bernett et al, 2002).…”