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.