L-thiocitrulline. A stereospecific, heme-binding inhibitor of nitric-oxide synthases.
Nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide (NO). The enzyme is inhibited by a variety of N omega-monosubstituted L-arginine analogs, and some of these compounds are useful in reversing pathologies associated with the overproduction of NO (e.g. the hypotension of septic shock). We report here that L-thiocitrulline (gamma-thioureido-L-norvaline) is a potent, stereospecific inhibitor of the constitutive brain and endothelial isoforms of NOS as well as the isoform induced in vascular smooth muscle cells by lipopolysaccharide and interferon-gamma. Steady state kinetic studies show L-thiocitrulline inhibition is competitive with L-arginine (Ki approximately 4-20% of KArgm), indicating that initial binding is as a substrate/product analog. In contrast to L-arginine and N omega-methyl-L-arginine, the prototypic NOS inhibitor, L-thiocitrulline binding elicits a "Type II" difference spectrum, indicating a high spin to low spin transition of the iron in the heme cofactor. This finding suggests that L-thiocitrulline is contributing the sixth ligand to heme iron, probably through the thioureido sulfur. Such interaction with heme iron neither stimulates nor inhibits the direct flavin-mediated cytochrome c reduction activity of the enzyme, but it does inhibit heme-dependent superoxide formation. In vivo, L-thiocitrulline is a potent pressor agent in both normal and endotoxemic rats, the latter finding suggesting utility in treating the hypotension of septic shock.
Frey
C
C
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Narayanan
K
K
McMillan
K
K
Spack
L
L
Gross
S S
SS
Masters
B S
BS
Griffith
O W
OW
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001665:Binding Sites; D002956:Citrulline / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D019001:Nitric Oxide Synthase; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D013890:Thiourea / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D019323:omega-N-Methylarginine
D003574:Cytochrome c Group; C086715:thiocitrulline; D019323:omega-N-Methylarginine; D002956:Citrulline; D006418:Heme; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D013890:Thiourea