Identification of residues critical for enzymatic activity in the domain encoded by exons 8 and 9 of the human inducible nitric oxide synthase.
Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of several diseases including airway inflammation of asthma. iNOS is active only as a homodimer. We previously demonstrated that the region encoded by exons 8 and 9 is critical for dimerization. In this study, alanine-scanning mutagenesis was used to identify critical amino acids in that region by expression of mutant proteins in human embryonic kidney 293 cells. All iNOS mutants yielded iNOS protein as detected by Western analysis. Four iNOS mutants with alanine replacing Trp260, Asn261, Tyr267, or Asp280 did not generate NO. Dimer formation was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis at 4 degrees C, followed by immunoblotting. Wild-type iNOS migrated both as monomers and dimers. iNOS mutants with alanine replacing Trp260, Asn261, or Tyr267, however, migrated only as monomers, suggesting that their inability to produce NO is related to a defect in dimer formation. Interestingly, the Asp280 mutant retained the ability to dimerize, indicating that it represents an inactive form of an iNOS dimer. These data identify four amino acids in exons 8 and 9 critical for iNOS activity, three of which also influence dimerization. These residues are strictly conserved among all NOS isforms and across species. Thus all NOS isoforms share general structural similarities, including specific amino acids critical for dimerization and catalytic activity. These data increase our understanding of the structural elements critical for NO synthesis and lay the groundwork for future studies aimed at downregulation of iNOS activity.
Eissa
N T
NT
Pulmonary and Critical Care Medicine Section, Department of Medicine, Baylor College of Medicine, Houston, Texas, USA. teissa@bcm.tmc.edu
Haggerty
C M
CM
Palmer
C D
CD
Patton
W
W
Moss
J
J
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000502:physiology; D015153:Blotting, Western; D002460:Cell Line; D017124:Conserved Sequence; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000502:physiology; D005091:Exons / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D017434:Protein Structure, Tertiary / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014162:Transfection
C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D000409:Alanine