Substrate Specificity of NO Synthases:  Detailed Comparison of l-Arginine, Homo-l-arginine, Their N^ω-Hydroxy Derivatives, and N^ω-Hydroxynor-l-arginine

Abstract

A detailed comparison of the oxidation of five compounds closely related to L-arginine (Arg) by purified recombinant neuronal and macrophage NO synthases (NOS I and NOS II) was performed. Homo-L-arginine (homo-Arg) is oxidized by both NOSs in the presence of NADPH with major formation of NO and homo-L-citrulline, with a molar ratio of close to 1, and minor formation of N omega-hydroxyhomo-L-arginine (homo-NOHA). Oxidation of homo-NOHA by the two NOSs also leads to NO and homocitrulline in a 1:1 molar ratio. On the contrary, N omega-hydroxynor-L-arginine (nor-NOHA) is a very poor substrate of NOS I and II, which fails to produce significant amounts of nitrite. The catalytic efficiency of both NOSs markedly decreases in the order Arg > NOHA > homo-Arg > homo-NOHA, as shown by the 20- and 10-fold decrease of kcat/Km observed for NOS I and NOS II, respectively, when comparing Arg to homo-NOHA. The greater loss of catalytic efficiency for homo-Arg, when compared to that for Arg, appears to occur at the first step (N-hydroxylation) of the reaction. In that regard, it is noteworthy that the Vm values for NOHA and homo-NOHA oxidation are very similar (about 1 and 2 micromol of NO min-1 mg of protein-1 for NOS I and II, respectively). In fact, lengthening of the Arg chain by one CH2 leads not only to markedly decreased kcat/Km but also to clear disturbances in NOS functioning. This is shown by a greater accumulation of the N omega-hydroxyguanidine intermediate (homo-NOHA:homocitrulline ratio between 0.2 and 0.4) and an increased consumption of NADPH for NO formation (between 2.0 and 2.6 mol of NADPH consumed for the formation of 1 mol of NO in the case of homo-Arg, instead of 1.5 mol in the case of Arg). Most of the above results could be interpreted by comparing the possible positionings of the various substrates relative to the two NOS active oxygen species which are believed to be responsible for the two steps of the reaction.

Helicobacter pylori Induces Macrophage Apoptosis by Activation of Arginase II

“…The iNOSspecific inhibitor, N-iminoethyl-L-lysine (L-NIL), and the arginase inhibitor and iNOS substrate, N -hydroxy-L-arginine (NOHA), were purchased from Alexis Biochemicals (San Diego, CA). The specific arginase inhibitor, N -hydroxy-nor-L-arginine (norNOHA), which is not a substrate for iNOS, was prepared as previously described (21). ␣-Difluoromethylornithine (DFMO), an ODC inhibitor, was obtained from Ilex Oncology (San Antonio, TX).…”

Arginase blockade protects against hepatic damage in warm ischemia-reperfusion

“…It is produced as an intermediary step when L-arginine is metabolized by the NOS enzymes and is also a direct substrate for NOS [14;15]. Recently, a more potent synthetic analogue of NOHA was identified, N-omega-hydroxy-nor-L-arginine (nor-NOHA) [15][16][17]. This compound increases L-arginine availability by inhibiting arginase activity, but does not directly increase NO or citrulline production as it is not a substrate for NOS [15].…”

“…Recently, a more potent synthetic analogue of NOHA was identified, N-omega-hydroxy-nor-L-arginine (nor-NOHA) [15][16][17]. This compound increases L-arginine availability by inhibiting arginase activity, but does not directly increase NO or citrulline production as it is not a substrate for NOS [15]. We chose to utilize this compound because of its low IC 50 and due to the fact that it does not directly interact as either a substrate or an inhibitor of NOS activity when compared to NOHA [15;18].…”

Liver I/R injury is improved by the arginase inhibitor, Nω-hydroxy-nor-l-arginine (nor-NOHA)

“…The naturally occurring arginase inhibitor is N -hydroxy-L-arginine (NOHA). It is produced as an intermediary step when L-arginine is metabolized by the NOS enzymes and is also a direct substrate for NOS (1,16). Recently, a more potent synthetic analog of NOHA was identified, N -hydroxynor-L-arginine (nor-NOHA) (5,16).…”