Oxidation of N^ω-Hydroxyarginine Analogues by NO-Synthase: The Simple, Non Amino Acid N-Butyl N‘-Hydroxyguanidine Is Almost as Efficient an NO Precursor as N^ω-Hydroxyarginine

Abstract

Introduction. Nitric oxide (NO) is a key inter-and intracellular messenger molecule involved in the maintenance of vascular tone, neuronal signaling, and host response to infection. 1,2 The biosynthesis of NO is catalyzed by constitutively expressed neuronal and endothelial nitric oxide synthases (NOS I and NOS III, respectively) and by inducible NOS (NOS II) that is expressed in macrophages following induction by inflammatory mediators. 2-5 All three NOSs produce NO and L-citrulline from the oxidation of L-arginine (L-arg) by NADPH and O 2 with formation of N ω -hydroxy-Larginine (NOHA) as an intermediate. 6,7 Because of the great importance of either an excess of NO or a deficit of NO in many physiopathological situations, 2-5 many groups have worked during these last years to find selective NOS inhibitors and substrates. Whereas a great number of strong NOS inhibitors have been described, 8 only a few NOS substrates have been reported so far. Most of them are R-amino acids closely related to L-arg or NOHA, such as homo-

Importance of valine 567 in substrate recognition and oxidation by neuronal nitric oxide synthase

“…Calmodulin, 2 0 ,5 0 -ADP-Sepharose, CaM-Sepharose and Sephadex G25 were products of Amersham-Pharmacia Biotech Inc. L L -Arg, NADPH, superoxide dismutase (SOD), catalase, hemoglobin, and other reagents were obtained from Sigma or Wako Pure Chemicals, and were of the highest purity commercially The synthesis and physico-chemical characteristics of NOHA [26], N -(iso-propyl)-N 0 -hydroxyguanidine 1 (iPr-NOHG), N -(n-butyl)-N 0 -hydroxyguanidine 2 (nBu-NOHG), N -(4-hydroxyphenyl)-N 0 -hydroxyguanidine 3 (OH-Ph-NOHG) have been published previously [21,22].…”

“…Hydrogen-bonds between the a-carboxylate group of L L -Arg and the hydroxyl group of Tyr588 in rat nNOS are also of importance for recognition of a-amino acids at the active site, and mutation of Tyr588 strongly alters the substrate specificity [19]. Recently, NOSs have been shown to catalyse NO-formation by oxidation of some non-amino acid alkylguanidines and N -alkyl-and N -aryl-N 0 -hydroxyguanidines following reactions similar to the oxidations of natural substrates, L L -Arg and NOHA [20][21][22]. The latest crystal structures reveal a new binding mode for two alternative substrates that accounts for their ability to be oxidised in nNOS active site [13].…”

Involvement of NO in the Endothelium-Independent Relaxing Effects ofNω-Hydroxy-l-arginine and Other Compounds Bearing a C=NOH Function in the Rat Aorta

“…4 and Table 1). The structural determinants required for the relaxant effect are also entirely different from those required for being an NO synthase substrate (Dijols et al, 2001;Renodon-Cornière et al, 2002): the substituted N-hydroxyguanidine, a poorly relaxant compound, is a selective substrate for the inducible NO synthase; by contrast, the identically substituted amidoxime ClBZA and ketoxime ClBK are potent relaxant compounds, whereas they are not substrate for any of the three NO synthase isoforms. Thus, structure activity relationships together with the above-mentioned failure of P450 and NO synthase inhibitors to blunt relaxation lend no support to the hypothesis of the involvement of a P450 or NO synthase in the relaxant effect of compounds with a CϭNOH function in the rat aorta.…”

OXIDATION OFN-HYDROXYGUANIDINES BY CYTOCHROMES P450 AND NO-SYNTHASES AND FORMATION OF NITRIC OXIDE

“…Deletion of either the a-COOH or the a-NH 2 function of NOHA led to compounds, N-hydroxy-agmatine and desamino-NOHA, respectively, that were much less active than NOHA (8 and 1% of the rate of NO formation from NOHA) ( Table 3). [20] However, quite surprisingly, deletion of both the a-COOH and a-NH 2 functions of NOHA led to a simple alkyl-N-hydroxyguanidine, N-butyl-N 0 -hydroxyguanidine, that was oxidized by NOS II with a rate of NO formation comparable to that observed in the case of NOHA (68%). [20] N-propyl-and N-pentyl-N 0 -hydroxyguanidine also act as reasonable substrates for NOS II, although they lead to lower rates of NO formation (Table 3).…”

“…[20] However, quite surprisingly, deletion of both the a-COOH and a-NH 2 functions of NOHA led to a simple alkyl-N-hydroxyguanidine, N-butyl-N 0 -hydroxyguanidine, that was oxidized by NOS II with a rate of NO formation comparable to that observed in the case of NOHA (68%). [20] N-propyl-and N-pentyl-N 0 -hydroxyguanidine also act as reasonable substrates for NOS II, although they lead to lower rates of NO formation (Table 3). Interestingly, N-hexyl-N 0 -hydroxyguanidine is almost inactive.…”