Studies of neuronal nitric oxide synthase inactivation by diverse suicide inhibitors.
N(G)-Amino-l-arginine, N(5)-(1-iminoethyl)-l-ornithine, N(6)-(1-iminoethyl)-l-lysine, and aminoguanidine were studied for the mechanisms by which they produce suicidal inactivation of the neuronal nitric oxide synthase isoform (nNOS). All of the inactivators that were amino acid structural analogs targeted the heme residue at the nNOS active site and led to its destruction as evidenced by the time- and concentration-dependent loss of the nNOS heme fluorescence, which reflects the disruption of the protoporphyrin-conjugated structure. The loss of heme was exclusively associated with the dimeric population of the nNOS. This inactivator-mediated loss of the nNOS heme never reached more than 60%, suggesting that only half of the dimeric heme is involved in catalytic activation of mechanism-based inactivators studied. Aminoguanidine-induced nNOS inactivation produced covalent modification of the nNOS protein chain with a stoichiometry of 0.8 mol of aminoguanidine per mole of the nNOS monomer. Specific covalent modification by aminoguanidine was exclusively associated with the oxygenase domain of the nNOS. The mechanisms by which N(6)-(1-iminoethyl)-l-lysine and aminoguanidine inactivate the nNOS and iNOS do not differ between the isoforms. The selectivity of these inactivators toward the iNOS isoform is a reflection of their much lower partition ratios, which were determined to be 0.16 +/- 0. 1 for N(6)-(1-iminoethyl)-l-lysine and 12 +/- 1.5 for aminoguanidine in case of the iNOS isoform while the same inactivators produced the partition ratios of 17 +/- 2 and 206 +/- 4, respectively, for the nNOS isoform.
Bryk
R
R
Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey, 08854, USA.
Lubeskie
A
A
Wolff
D J
DJ
D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D020134:Catalytic Domain / Q000187:drug effects*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D006146:Guanidines / Q000494:pharmacology; D006420:Hemeproteins / Q000187:drug effects*; D008239:Lysine / Q000031:analogs & derivatives / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000187:drug effects; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D009952:Ornithine / Q000031:analogs & derivatives / Q000494:pharmacology; D020836:Protein Structure, Quaternary
D006146:Guanidines; D006420:Hemeproteins; C090663:N(6)-(1-iminoethyl)lysine; C065027:N(G)-iminoethylornithine; C063904:N(G)-aminoarginine; D001120:Arginine; D009952:Ornithine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D008239:Lysine; C004479:pimagedine