Relationship between the structure of guanidines and N‐hydroxyguanidines, their binding to inducible nitric oxide synthase (iNOS) and their iNOS‐catalysed oxidation to NO

The binding of several alkyl- and aryl-guanidines and N-hydroxyguanidines to the oxygenase domain of inducible NO-synthase (iNOS(oxy)) was studied by UV/Vis difference spectroscopy. In a very general manner, monosubstituted guanidines exhibited affinities for iNOS(oxy) that were very close to those of the corresponding N-hydroxyguanidines. The highest affinities were observed for the natural substrates, L-arginine and N(omega)-hydroxy-L-arginine (K(d) at the microm level). The deletion of either the CO2H or the NH2 function of their amino acid moiety led to dramatic decreases in the affinity. However, alkylguanidines with a relatively small alkyl chain exhibited interesting affinities, the best being observed for a butyl chain (K(d) =20 microM). Arylguanidines also bound to iNOS(oxy), however, with lower affinities (K(d) > 250 microm). Many N-alkyl- and N-aryl-N'-hydroxyguanidines are oxidized by iNOS with formation of NO, whereas only few alkylguanidines led to significant production of NO under identical conditions, and all the arylguanidines tested to date were unable to lead to the production of NO. The k(cat) values of NO production from the oxidation by iNOS of the studied N-hydroxyguanidines were found to vary independently of their affinity for the protein. The k(cat) values determined for the two-step oxidation of alkylguanidines to NO were not clearly related to the K(d) of these substrates toward iNOS(oxy). However, there is a qualitative relationship between these k(cat) values and the apparent rate constants of dissociation of the complex between iNOS(oxy) and the corresponding N-alkyl-N'-hydroxyguanidine (k(off) (app)) that were determined by stopped-flow UV/Vis spectroscopy. These data indicate that a key factor for efficient oxidation of a guanidine by iNOS to NO is the ability of the corresponding N-hydroxyguanidine to bind to the active site without being too rapidly released before its further oxidation. This explains why 4,4,4-trifluorobutylguanidine is so far the best non-alpha-amino acid guanidine substrate of iNOS with formation of NO, because the k(off) (app) of the corresponding N-hydroxyguanidine is particularly low. This suggests that the rational design of guanidines as new NO donors upon in situ oxidation by NOSs should take into account both thermodynamic and kinetic characteristics of the interaction of the protein not only with the guanidine but also with the corresponding N-hydroxyguanidine.