Neuronal nitric oxide synthase. Expression in Escherichia coli, irreversible inhibition by phenyldiazene, and active site topology.
A gene coding for rat neuronal nitric oxide synthase (nNOS) has been cloned into pCWori and the vector has been expressed in Escherichia coli. The expressed enzyme has been purified with a final yield of purified protein of approximately 1 mg/g of wet cells. The recombinant protein reconstituted with calmodulin and Ca2+ exhibits spectroscopic and catalytic properties identical to those reported in the literature for nNOS. Reaction of recombinant nNOS with phenyldiazene produces a phenyl-iron (Fe.Ph) complex with a maximum at 470 nm. Formation of this complex is paralleled by inactivation of the enzyme and is inhibited by arginine, the natural substrate of the enzyme. Phenyl-iron complex formation does not alter the rate of electron transfer from the flavin domain to cytochrome c. Addition of ferricyanide triggers migration of the phenyl residue from the iron to the porphyrin nitrogens. The N-phenylprotoporphyrin isomers with the phenyl on the nitrogens of pyrrole rings B, A, C, and D are formed in, respectively, approximately a 14:20:21:45 ratio. The regioisomer pattern indicates that the active site of NOS is open to some extent above all four pyrrole rings but more so above pyrrole ring D. Arylhydrazines are thus not only a new class of inhibitors of nNOS but provide useful information on the active site topology of the enzyme.
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Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Ortiz de Montellano
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D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D007097:Imines / Q000494:pharmacology*; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase; D011487:Protein Conformation
D007097:Imines; C011981:phenyldiazene; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases