Novel substrates for nitric oxide synthases.

Enzymatic generation of nitric oxide (NO) by nitric oxide synthase (NOS) consists of two oxidation steps. The first step converts L-arginine to N(G)-hydroxy-L-arginine (NOHA), a key intermediate, and the second step converts NOHA to NO and L-citrulline. To fully probe the substrate specificity of the second enzymatic step, an extensive structural screening was carried out using a series of N-alkyl (and N-aryl) substituted-N'-hydroxyguanidines (1-14). Among the eleven N-alkyl-N'-hydroxyguanidines evaluated, N-n-propyl (2), N-iso-propyl (3), N-n-butyl (4), N-s-butyl (5), N-iso-butyl (6), N-pentyl (8) and N-iso-pentyl (9) derivatives were efficiently oxidized by the three isoenzymes of NOS (nNOS, iNOS and eNOS) to generate NO. N-Butyl-N'-hydroxyguanidine (4) was the best substrate for iNOS (K(m)=33 microM) and N-iso-propyl-N'-hydroxyguanidine (3) was the best substrate for nNOS (K(m)=56 microM). When the alkyl substituents were too small (such as ethyl 1) or too large (such as hexyl 10 and cyclohexyl 11), the activity decreased significantly. This suggests that the van der Waals interaction between the alkyl group and the hydrophobic cavity in the NOS active site contributes significantly to the relative reactivity of compounds 3-11. Moreover, five N-aryl-N'-hydroxyguanidines were found to be good substrates for iNOS, but not substrates for eNOS and nNOS. N-phenyl-N'-hydroxyguanidine was the best substrate among them (K(m)=243 microM). This work demonstrates that N-alkyl substituted hydroxyguanidine compounds are novel NOS substrates which 'short-circuit' the first oxidation step of NOS, and N-aryl substituted hydroxyguanidine compounds are isoform selective NOS substrate.